Xia Chun-Fang, Boado Ruben J, Pardridge William M
Department of Medicine, UCLA, Los Angeles, California 90024, USA.
Mol Pharm. 2009 May-Jun;6(3):747-51. doi: 10.1021/mp800194y.
Delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. In the present studies, the luciferase gene is transiently expressed in human 293 epithelial cells. The siRNA delivery system is composed of the siRNA, monobiotinylated on the 3'-terminus of the sense strand, and a conjugate of streptavidin (SA) and a mAb to the human insulin receptor (HIR). Exposure of cells to 3'-biotinyl-siRNA bound to the HIRMAb/SA conjugate, but not to unconjugated SA, avidin, or the HIRMAb, causes a >90% reduction in luciferase gene expression. The receptor-targeted siRNA effect is maximal at 48 h after delivery of the siRNA to the cells, and the effect is lost by 7 days after a single application of the targeted siRNA in culture. The KI of the receptor-targeted siRNA inhibition of gene expression is 30.5 +/- 11.7 nM, and significant inhibition is observed with siRNA concentrations as low as 3 nM. In conclusion, the combination of a receptor-specific targeting ligand, such as the HIRMAb, and avidin-biotin technology allows for high affinity capture of the monobiotinylated siRNA by the targeting mAb. The siRNA is effectively delivered to the cytosol of cells, and knockdown of gene expression with the HIRMAb/SA delivery system is comparable to RNA interference effects obtained with cationic polyplexes. Whereas the use of cationic polyplexes in vivo is problematic, the bond between the targeting mAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA.
通过联合使用受体特异性单克隆抗体（mAb）和抗生物素蛋白-生物素技术，可将小干扰RNA（siRNA）递送至培养细胞及体内细胞。在本研究中，荧光素酶基因在人293上皮细胞中瞬时表达。siRNA递送系统由在正义链3'-末端单生物素化的siRNA以及链霉亲和素（SA）与抗人胰岛素受体（HIR）的mAb的缀合物组成。将细胞暴露于与HIRMAb/SA缀合物结合的3'-生物素化-siRNA，而非未缀合的SA、抗生物素蛋白或HIRMAb，会导致荧光素酶基因表达降低>90%。受体靶向siRNA的作用在将siRNA递送至细胞后48小时达到最大，且在培养中单次应用靶向siRNA后7天作用消失。受体靶向siRNA抑制基因表达的解离常数（KI）为30.5±11.7 nM，在低至3 nM的siRNA浓度下即可观察到显著抑制。总之，受体特异性靶向配体（如HIRMAb）与抗生物素蛋白-生物素技术的结合使得靶向mAb能够高亲和力捕获单生物素化的siRNA。siRNA可有效递送至细胞胞质溶胶，并且使用HIRMAb/SA递送系统敲低基因表达与阳离子多聚体获得的RNA干扰效果相当。虽然在体内使用阳离子多聚体存在问题，但靶向mAb与siRNA之间的结合通过抗生物素蛋白-生物素技术是稳定的，并且在静脉内给予靶向siRNA后，在体内可观察到在诸如脑等远处部位的RNAi效应。
