We have proven the utility of this approach by designing and testing a specific inhibitor for TNF a . An in vitro translation system was employed to determine whether the association between padlock RNA and the target mRNA was sufficient to inhibit translation. The hybrid mRNA was incubated with padlock RNA, or antisense RNA to allow binding to occur, and then placed into rabbit reticulocyte lysate for in vitro translation. Hybrid mRNA was efficiently translated into luciferase protein when incubated with antisense RNA, but translation was reduced by The single greatest difficulty for nucleic acid based therapeutics or gene therapy is delivery of the nucleic acid into mammalian cells. Dose response curves for the three newer ATR-type constructs directed against three different target sequences of TNFa mRNA in cultured cells.
Error bars show standard deviation of the mean.
Figure 1 illustrates that macrophages not incubated with padlock RNA, or incubated with padlock RNA not targeted to TNF a produced similar, high levels of TNF a protein. Three of the four padlock RNA constructs were effective inhibitors of TNFa secretion by macrophages, indicating that the design parameters employed were predictive of efficacy. We have demonstrated that nucleic acids can be efficiently delivered to macrophages and tissues of the reticuloendothelial system in vivo . RNA after administration to mice via the trachea. Many of the cells lining the alveoli, including cells morphologically consistent with alveolar macrophages accumulated padlock RNA administered as a complex with cationic lipid.