Figure 8: hmgcr RNAi using DY-635[NP] as carrier.

(a) A significant reduction in hmgcr gene-expression was achieved with a maximum at 16 h after transfection in naive Hepa1-6 cells. In contrast to DY-635[NP](siRNA) an increase in hmgcr was observed after application of the carrier (DY-635[NP](siCtrl)). Mean±s.e.m. *P<0.05 compared with respective siCtrl by ANOVA and Tukey post hoc. (b,c) To validate siRNAs, the efficacy of two RNAi sequences (shRNA#1, shRNA#2) against hmgcr was analysed in a heterologous expression model using western blot (b), and addressed by determining endogenous HMGCR levels in HepG2 cells using anti-HMGCR immuno-fluorescence analysis (c). A significant reduction in hmgcr protein could be achieved with both RNAis; for further RNAi experiments RNAi sequence#2 was selected. Mean±s.e.m. *P<0.05 versus control by ANOVA and Dunnett’s test. (d) In vivo RNAi results in mice injected with [NP](siHMGCR) (n=9) or DY-635[NP] loaded with siHMGCR (n=12) or a control RNA (siCtrl) (n=12); animals injected with the vehicle (5% sterile glucose solution; n=4) served as sham controls. hmgcr gene expression was analysed using relative quantification compared with untreated anminals (n=4) in RNA prepared from liver tissue by RT-qPCR. Consistent with the results of the cell culture, DY-635[NP](siCtrl) induced the hmgcr gene expression while [NP](siHMGCR) blunted this effect but failed to lower the hmgcr expression compared with untreated or sham animals. Using similar siHMGCR amounts encapsulated in DY-635[NP](siHMGCR), hmgcr expression was lowered by 75% compared with untreated animals. Mean±s.e.m. *P<0.05 by ANOVA and Tukey test. (e) Altered gene expression of hmgcr was reflected in plasma-cholesterol levels of these animals. Mean±s.e.m. by ANOVA and Tukey test.