Inhibition of tumor necrosis factor alpha secretion by a 27‐mer siRNA in rat Kupffer cells

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. Earlier studies show that two daily doses (1.5 mg/kg body wt.) of an antisense oligonucleotide against rat TNF‐α mRNA, reduced lipopolysaccharide (LPS)‐induced liver injury by 60%. 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 may have much higher efficacies. In this study, we report the identification of a 27‐mer anti‐TNF‐α siRNA and compare its properties with a recently identified 21‐mer anti‐TNF‐α siRNA, SSL3. Primary cells of rat Kupffer cells were transfected with 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 ug/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 5 nM, si27‐3, almost totally (>97%) inhibited TNF‐α secretion compared to an 85% inhibition by the 21‐mer (SSL3); at 1 nM, si27‐3 was still inhibitory at 95% after 48 h whereas the inhibitory effect of SSL3 was lower at 75%. These data demonstrate the identification of a highly efficacious siRNA, which can be used in the treatment of TNF‐α mediated diseases (Supported by Grant AA 015081).