Preliminary Study Of Asymmetric Sirna Targeting Ccr5,Bcl2and Vegf Gene

Fire and Mello founded RNA interference theory in1998, then the study of RNA interference (RNAi) became more and more popular, Continued emergence of new research results. Small interfering RNA (siRNA) has emerged as a new type of therapy, fifteen of the siRNA drugs have entered Ⅰ to Ⅲ clinical research stage. In2008, Sun and some of the other scientists proposed the asymmetry small interfering RNA (asiRNA) in mammalian cells can lead to more effective silencing effect of target gene and can reduce off-target effects. Therefore, the optimized asiRNA may have potential as a specific and safe RNAi therapeutic.We designed a series of asymmetric shorter-duplex siRNAs (asiRNAs) targeting bcl2, VEGF and CCR5, and a proteolipid micelle was used to deliver asiRNA targeting bcl2, VEGF or CCR5. The proteolipid micelle (named PDE) composed of polyethylene glycol-phosphatidylethanolamine (PEG-PE), phospholipid dioleylphosphatidylserine (DOPS) and rhEndostatin.The in vivo fluorescence imaging assay was used of Cy5fluorescence labeled CCR5-asiRNA (CyasiRNA). The ELISA-based assay was used of dual-labeled CCR5-asiRNA (QasiRNA),5’-end-labeled on one strand with biotin, and with dinitrophenol (DNP) on the other end. The results demonstrated that modification of asiRNA can improve the stability of asiRNA in serum of appropriate condition. The results showed the higher distribution of QasiRNA in the blood, liver, lung and kidney at15min after administration using the ELISA-based assay, and the highest distribution of CyasiRNA in the kidney at24hour after administration using the in vivo fluorescence imaging assay. Moreover, both methods revealed the lowest levels of QasiRNA or CyasiRNA in the spleen. These results demonstrate that both of the two methods are accurate, sensitive, convenient method for asiRNA targeting CCR5delivered via cationic liposome after intravenous administration, particularly from a pharmacokinetic perspective.Furthermore, in in vivo experiments, the results demonstrated the asiRNA targeting bcl2or VEGF could efficiently suppress the growth of BGC-803tumors in vivo. These results suggest that asiRNAs may have potential as an effective approach for cancer gene therapy in the future.