Silencing Efficiency And Off-target Effect Of Asymmetric SiRNA Targeting Bcl-2Gene

Small interfering RNAs (siRNAs) are valuable reagents for efficient gene silencing in a sequence-specific manner via the RNA interference (RNAi) pathway. The current synthetic siRNA structure consists of symmetrical duplexes of19-21base pairs (bp) with2nucleotide (nt)3’overhangs. However, this siRNA structure triggers several non-specific effects posing challenges to the application of RNAi therapeutics in clinical practice. Recent studies have shown that the asymmetric siRNAs (asiRNAs) were capable of effectively silencing target gene expressions and caused less off-target effects. In the present study, we synthesized a series of asiRNAs ranging from the13-17bp duplex region (13-17bp asiRNAs) targeting bcl-2gene, and evaluated their silencing ability and off-target effects in vitro. The results showed that17bp asiRNA had potent activity in downregulating bcl-2gene expression and inhibiting tumor proliferation in vitro. Importantly, this asiRNA structure significantly reduced off-target effects compared with conventional siRNA. Furthermore,17bp asiRNA targeting bcl-2gene could reverse multidrug resistance of tumor cells.The poor stability and inefficient delivery of siRNA are two major obstacles for the therapeutic application of siRNA following systemic administration. Chemical modification is an effective method for improving the stability of siRNA and several studies have reported that some siRNAs with chemical modifications show durable activities in RNAi assays. To improve the stability of the17bp asiRNA, we introduced a series of chemically modification to17bp asiRNAs, including phosphorothioate,2’-O-methylation (2’-O-Me),2’-fluoro-uridine (2’-FU) and cholesterol conjunction. The comparison of silencing effect suggested that chemical modifications reduced the silencing activity of17bp asiRNA. The17bp asiRNA-M2, one of the modified17bp asiRNAs, exhibited the best gene silencing effect among all the chemically modified asiRNAs and its serum stability was significantly increased compared with17bp asiRNA. To further use asiRNA in the in vivo experiments, a proteolipid micelle was preparation to deliver asiRNA. The proteolipid micelle (termed PDE) was composed of polyethylene glycol-phosphatidylethanolamine (PEG-PE), phospholipid dioleylphosphatidylserine (DOPS) and rh-endostatin. The PDE was mixed with17bp asiRNA-M2to form17bp asiRNA-M2/PDE complexes, and its characterization and serum stability were studied. The results showed that the PDE micelle and17bp asiRNA-M2could form nano-size micelles with positive charge, and PDE micelle could protect17bp asiRNA-M2from nuclease in serum. The in vivo experiments suggested that17bp asiRNA/PDE complex could stably exist in mice for a long time and it effectively suppressed the growth of several tumor models in mice. The acute toxicity experiments showed that17bp asiRNA-M2/PDE complexes had no toxic effects.In conclusion, our study indicates that symmetrical duplex is not necessary for the silencing effect of siRNA, and asymmetric siRNAs can also effectively silence target gene while reducing off-target effects. The chemically modified17bp asiRNA-M2maintained silencing effect andr the serum stability of it was increased. When17bp asiRNA-M2was delivered by PDE micelle it is very stable and significantly inhibited tumor growth in vivo. Our results provide foundation for siRNA design and siRNA application in cancer therapy in the future.