title: Dicer-substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro: in vivo targeting of Kupffer cells by siRNA-liposomes.
abstract: 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 small interfering RNAs (siRNAs) to reduce TNF-α. Although siRNAs of 19-21bp are commonly used, it is reported that longer siRNAs have much higher efficacies. Here, we report the identification of a 27-mer Dicer-substrate siRNA (DsiRNA) against TNF-α mRNA. Primary cells of rat Kupffer cells were transfected with five 27-mer siRNA constructs (si27-1, si27-2 si27-3, si27-4 and si27-5) for 24h, following which, TNF-α secretion was induced by exposure to LPS (0.1μg/ml) for 2h. TNF-α released to the medium was measured by ELISA. Of the five si27 constructs, si27-3 had the highest inhibitory effect on TNF-α secretion. At 10nM, si27-3 inhibited TNF-α secretion by 80% compared to a 60% inhibition by a 21-mer (SSL3). Following encapsulation in anionic liposomes, si27-3 at 100μg/kg body weight, on two successive days by intravenous administration, inhibited the secretion of TNF-α by 50%. These data demonstrate the identification of a highly efficacious siRNA formulation, which can be used in the treatment of TNF-α mediated diseases.
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, United States.
authors.forename: Sivakamasundari
authors.initials: S
authors.lastname: Pichu
authors.forename: Swapna
authors.initials: S
authors.lastname: Krishnamoorthy
authors.forename: Bi
authors.initials: B
authors.lastname: Zhang
authors.forename: Yawu
authors.initials: Y
authors.lastname: Jing
authors.forename: Andrei
authors.initials: A
authors.lastname: Shishkov
authors.forename: Biddanda C
authors.initials: BC
authors.lastname: Ponnappa
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D015536:Down-Regulation; D015316:Genetic Therapy / Q000379:methods*; D007275:Injections, Intravenous; D007728:Kupffer Cells / Q000187:drug effects / Q000201:enzymology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008081:Liposomes; D008297:Male; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043244:Ribonuclease III / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000097:blood / Q000235:genetics / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D008081:Liposomes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D043244:Ribonuclease III