Purpose: We investigated if an anti-TNF-α triplex-forming oligonucleotide (TFO), employed as anti-gene strategy, could be an alternative to antisense technology in silencing TNF-α activation during articular inflammation. In this study, we analyzed in vitro and in vivo anti-inflammatory potentialities of an anti-TNF-α TFO, as judged from effects on two rat arthritis models. Methods: The inhibitory activity of this TFO on articular cells stimulated by IL-1ß (synoviocytes and chondrocytes) was assessed and compared to that of small interfering RNA (siRNA) in vitro, at mRNA expression levels by real-time RT-PCR, as well as at proteins level (TNF-α & NO release). In vivo, the biological effects of a preventive intraarticular injection of such an oligonucleotide were first investigated in an acute arthritis model, by the follow-up of clinical (body weight, pain, joint swelling), biochemical (anabolism loss, cytokines levels) and histological (synovium, cartilage,and bone) parameters. We then confirmed the efficiency of a preventive ia injection of anti-TNF-α TFO in an immunological experimental chronic arthritis model, according to similar parameters (clinical, biochemical and histological assessments). Results: In vitro, we have demonstrated that a TFO designed to target TNF-α promoter was able to inhibit mRNA expression (86%) and to prevent TNF-α release into supernatants (62%) as well as NO release (80%). The inhibition rate, at mRNA level, was similar to the one observed with TNF-α specific siRNA, the main advantage of TFO being the concentration used (1nM vs 75 nM for siRNA). A difference was observed at proteins level, with approximatively a 20% more efficient inhibition with TFO, compared to siRNA. The use of the anti-TNF-α TFO as a preventive and local treatment in both acute and chronic arthritis models significantly reduced disease development. In the acute inflammatory model, the TFO provided a stronger inhibition than the siRNA, with a long lasting biological effect, confirmed by histological assessement. In the immunological RA model, we have demonstrated that a preventive ia injection of the TFO lead to a significant correction of body weight distribution (incapacitancy test), confirmed by the histological grading of the knees. Indeed, fibrosis and infiltration were significantly decreased compraed to arthritic rats. Analysis of synovial fluid has demonstrated a significant decrease of inflammatory mediators like cytokines (TNF-a, IL-1ß, IL-2, IL-5) and chemokines (MCP1, MIP-1a) in animals preventively treated with TFO compared to naives. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. Conclusions: We showed the effectiveness of siRNA and TFO in modulating both in vitro and in vivo inflammatory processes. Interestingly, silencing was increased with TFO, enabling improved protection of articular components. We extended our findings by demonstrating for the first time that in rats developing arthritis, a preventive injection of anti-TNF-αTFO led to local and systemic TNF-α inhibition associated with improvement of ancillary clinical signs of arthritis. The results presented here provide the first evidence that gene targeting by anti-TNF-α TFO modulates arthritis in vivo, thus providing proof-of-concept that it could be used as therapeutic tool for TNF-α-dependent inflammatory disorders.