Dicer‐substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro and in vivo

Tumor necrosis factor alpha (TNF‐α) plays a major role in the pathogenesis of many inflammatory diseases. Neutralizing TNF‐α by antibodies or antisense oligodeoxynucleotides, alleviate disease symptoms. In this study, we introduce the new generation of gene‐silencing molecules, namely the short interfering RNA (siRNA), to reduce TNF‐α levels. Although siRNAs of 19–21 base pairs are commonly used, it is reported that longer siRNAs have much higher efficacies. In this study, we report the identification of a 27‐mer anti‐TNF‐α siRNA. Primary cells of rat Kupffer cells were transfected with one of three 27‐mer siRNA constructs (si27‐1, si27‐2 and si27‐3) for 24 h, following which, TNF‐α secretion was induced by exposure to LPS (0.1 μg/ml) for 2 h. TNF‐α released to the medium was measured by ELISA. Of the three si27 constructs, two (si27‐2 and si27‐3) had profound inhibitory effect on TNF‐α secretion and the third had no effect at all. At 10 nM concentration, si27‐3 inhibited TNF‐α secretion by 80%, compared to a 60% inhibition by the 21‐mer (SSL3). Following encapsulation in anion liposomes, intravenous administration of si27‐3 (100 μg/kg/body wt) on two successive days inhibited the secretion of TNF‐α by 40–50%. These data demonstrate the identification of a highly efficacious siRNA formulation which can be used in the treatment of TNF‐α mediated diseases (Supported by NIH Grant AA016551).