Résumé
Background: TNF is a key cytokine in rheumatoid arthritis (RA). Using a new liposome formulation we showed that intravenous injection of a small interfering RNA (siRNA) anti-TNF efficiently restored the immunological balance in mice with collagen-induced arthritis (CIA). We took advantage of our simple RNAi-based screening system to investigate which cytokine other than TNF could be targeted for therapeutic benefit in the 30% of patients that do not respond to anti-TNF biotherapies. Methods: The siRNA sequences against IL-1, IL-6 and IL-18 were validated in vitro on macrophage cell lines, at mRNA and protein levels after LPS challenge. For in vivo screening, siR-NAs were formulated as lipoplexes with the RPR209120/ DOPE liposome and a carrier DNA, and injected intravenously once a week in CIA mice (0.5 mg/kg). Paw thickness was assessed over time, and radiological and histological scores were obtained at euthanasia. The cytokine profiles were measured by ELISA in sera and knee-conditioned media. The anti-type II collagen antibodies were detected by ELISA. The best anti-cytokine siRNA was then cloned in a recombinant adeno-associated serotype 5 virus (rAAV5) for local intra-articular delivery to CIA mice (5 x 109 vp/knee). Results: The designed siRNA sequences silenced specifically 70-75% of the LPS-induced IL-1, IL-6 and IL-18 mRNA expression in macrophages compared with a control siRNA. In the CIA model, a weekly injection of siRNA-lipoplexes abrogates joint swelling, destruction of cartilage and bone, in both preventive and curative settings. The 3 anti-cytokine siRNA lipoplexes were effective to different extend, and the most striking therapeutic effect was observed when combining the 3 siRNAs targeting IL-1/IL-6/IL-18 at once. Such tri-therapy was associated with down-regulation of both inflammatory and autoimmune components of the disease, and overall parameters were improved compared with the TNF siRNA lipoplex-based treatment. A single injection of rAAV5-shRNA-TNF allowed stabilizing clinical features locally. Conclusion: The systemic administration of siRNA lipoplexes represents a useful screening tool for new therapeutic candidates that can be further expressed by a rAAV5 vector for long term targeted treatment in RA.