Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA

Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. However, the concern about the specificity of present RNA-knockdown strategies has limited their in vivo applications. Here a TaqTth-hpRNA system consisting of a small, chimeric protein (TaqTth) and hairpin-RNA probe (hpRNA) is provided. The TaqTth-hpRNA showed a high-specific knockdown against targeted mRNA with minimal flanking sequence-motif requirement and less cell viability damage, then was applied to mutant APPswe mRNA silencing without altering the wild-type APP mRNA in Alzheimer’s disease. Notably, the combination of the TaqTth and human apolipoprotein E 2 (APOE2) overexpression encoded in a single AAV vector is available due to the compact size of TaqTth, and showed stronger inhibition of pathologies in the mouse model. Altogether, we provided the basis for a small RNA-targeting tool with high selectivity, minimal flanking sequence-motif requirement, less cell viability damage and potentially being an alternative in therapeutic applications. To examine the intensity of the collateral effects of the TaqTth-hpRNA, we performed total RNA integrity analysis and transcriptome-wide RNA-seq of HepG2 cells treated with TaqTth-hpRNA or siRNA. Similar to previous findings , a significant reduction in RNA integrity was observed in the siRNA-treated cells. We also observed off-target degradation of transcripts in cells treated with PPIB siRNA. Data showed that ~ 1084 and 1110 genes were significantly up-regulated and down-regulated in siRNA group compared with the NT control, respectively . Further analysis showed that these off-targeted genes were distributed in many biological processes, including cell growth, cell locomotion, metabolic process and so on . These results are consistent with previous reports that host transcript degradation induced by siRNA results in retarded growth of cells. Unlike siRNA treatment, the TaqTth-hpRNA sharply reduced the number of off-target genes when targeting PPIB (26 vs 2194) , indicating that TaqTth mainly reduced collateral off-target degradation.