Objective. Tumor necrosis factor ␣ (TNF␣) is among the most prominent cytokines in rheumatoid arthritis (RA) and is secreted mainly by macrophages. A direct method for restoring the immunologic balance in RA is use of small interfering RNA (siRNA) for silencing the TNF␣ transcript. The aim of this study was to determine the therapeutic effect of systemic administration of TNF␣ siRNA in an experimental model of RA, optimizing its delivery using new liposome formulations.Methods. Murine macrophages were transfected with siRNA targeting TNF␣, and expression was measured. The therapeutic effect in collagen-induced arthritis (CIA) was assessed after intravenous delivery of TNF␣ siRNA. Delivery was optimized using a carrier DNA for complexation with the cationic liposome RPR209120/DOPE. Levels of TNF␣ and other cytokines were measured in sera and joint tissue-conditioned media. Biodistribution was determined using a fluorescent siRNA.Results. In vitro, TNF␣ siRNA efficiently and specifically modulated the expression of TNF␣ at both the messenger RNA and protein levels. In vivo, complete cure of CIA was observed when TNF␣ siRNA was administered weekly, complexed with the liposome and combined with carrier DNA. Inhibition (50-70%) of articular and systemic TNF␣ secretion was detected in the siRNA-injected groups, which correlated with a decrease in the levels of interleukin-6 and monocyte chemotactic protein 1. The main organs targeted by siRNA were the liver and spleen; the addition of liposome RPR209120 and carrier DNA significantly increased organ uptake.Conclusion. We demonstrated the efficiency of systemic delivery of siRNA designed to silence TNF␣ in CIA, using a liposome carrier system as a way to address the methodologic limitations in vivo.

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Objective. Blocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi)-based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA).Methods. Two synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA.Results. Weekly injections of anti-IL-1, anti-IL-6, or anti-IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex-based treatment. Conclusion.Our results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates.