Vector-Based in Vivo RNA Interference:Dose- and Time-Dependent Suppression of Transgene Expression

摘要

RNA interference (RNAi) induced by delivery of a small-interfering RNA (siRNA)-expressing vector was characterized in mice.SiRNA-expressing plasmid DNA (pDNA) was injected by a hydrodynamics-based procedure along with pDNA encoding an exogenous target luciferase gene.A comparative study showed that stem-loop-type siRNA-expressing pDNA was superior,in terms of the transgene suppressive efficacy,to the tandem-type in the liver following systemic delivery of these pDNAs.Transgene suppression occurred in the liver,kidney,and lung as well as muscle.The degree of suppression was dependent on the dose of siRNA-expressing pDNA and the timea t which transgene expression was determined following simultaneous injection of SiRNA-expressing and target pDNAs.A reduction in transgene expression became apparent at 1 day after injection,whereas a lower degree of inhibition was obtained before this,as early as 6 h even in mice treated with an excess of siRNA-expressing pDNA.These results suggest that delivery of siRNA-expressing pDNA requires a period of time for induction of RNAi.A study of sequential injections revealed that prior injection of siRNA-expressing pDNA produced a significant suppression for at least 1 day,which disappeared within 4 days.Confocal microscopic studies indicated that the localization of the cells with successful delivery of transgene was different between primary and secondary hydrodynamics-based injections,accounting for the less effective inhibition following the sequential injections.Taken together,these results demonstrated that vector-based in vivo RNAi is a dose- and time-dependent process and offers the possibility of suppressing endogenous targets in a variety of somatic cells.