Inhibition of tumor necrosis factor alpha secretion in rat Kupffer cells by siRNA: In vivo efficacy of siRNA-liposomes

Abstract

SummaryTumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease. In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-α by anti-TNF-α antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-α. The efficacies of various siRNA duplexes were tested against TNF-α mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-α by 55% (p<0.005) with a proportionate reduction in TNF-α mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 μg/kg body weight/day for two days) of liposome-encapsulated SSL3. The LPSinduced TNF-α secretion was inhibited by > 60% (p < 0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-α, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-α.

Dicer-substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro: In vivo targeting of Kupffer cells by siRNA-liposomes

“…We observed that si27-3 inhibited LPS-induced TNF-α secretion by 50% (Fig. 8), which is slightly better than the in vivo efficacy of SSL3, the 21-mer siRNA, which showed similar efficacy at a 40% higher concentration [29]. Our studies also show that siRNAs or DsiRNAs targeted against TNF-α are at least ten times more potent than the anti-TNF-α AsODNs used in an earlier study [19].…”

“…Having successfully used the liposomal delivery system to down-regulate TNF-α using AsODNs, it was our objective to identify highly effective siRNAs against TNF-α, so as to be able to achieve in vivo efficacy at as low a concentration of the nucleotide as possible. To that end, in a recent study [29] using a 21-mer siRNA (SSL3) against TNF-α and the same liposomal delivery system, we demonstrated that siRNA was an order of magnitude more sensitive than the single stranded anti-TNF-α AsODN [29]. The observation that Dicer-substrate siRNAs are even more potent than the traditional 21-mer siRNAs [30,31], gave us the opportunity to explore the therapeutic potential of DsiRNAs against TNF-α using the liposomal delivery system.…”

“…While the choice of the delivery system depends upon the target, it is generally observed that delivery of oligonucleotides in the ‘naked’ form is the least efficient one. In a recent study, we reported the identification of a 21-mer siRNA duplex, SSL3, against rat TNF-α mRNA, and its successful delivery in vivo using an anionic liposomal delivery system [29]. Although for gene-silencing studies, most researchers employ synthetic RNA duplexes that are 19–21 bases long, in 2005, it was demonstrated that the use of a slightly longer siRNAs, which are substrates for the Dicer enzyme, show higher potency than the traditional 21-mer siRNAs [30, 31].…”

Non-viral gene delivery carrier and its three-dimensional transfection system

“…This natural tendency for lung and liver uptake via intravenous administration may be exploited for eradication of pulmonary and hepatic metastases. Such an approach has been taken by several groups with varying degrees of success for pulmonary (Li et al, 2008b(Li et al, , 2005 and hepatic metastases (Zimmermann et al, 2006;Jing et al, 2008;Sato et al, 2007). The surfaces of liposomes always need to be modified by conjugating ligand or antibodies for targeting the specific cell types in the liver (Ko et al, 2009).…”

Effect of Streptomyces 23-2B metabolites on hepatic lipid peroxidation and some antioxidant parameters in Wister rats

“…In the liver, Kupffer cells are the major producers of TNF-a following exposure to bacterial endotoxin (Decker 1990). An overproduction of TNF-a has been associated with the development of alcoholic liver injury, rheumatoid arthritis, inflammatory bowel disease and septic shock (Jing et al 2008). Moreover, there is increasing evidence that nitric oxide (NO) can function as a cellular antioxidant and can, under certain conditions, protect against reactive oxygen species-induced toxicity (Wink et al 1995).…”