Combined anti-inflammatory tritherapy using a novel small interfering RNA lipoplex successfully prevents and cures mice of arthritis

Background

TNFα is a key cytokine in rheumatoid arthritis (RA) physiopathology. We recently demonstrated that a new cationic liposome formulation allowed intravenous delivery of a small interfering RNA (siRNA) targeting TNFα and efficiently restoring the immunological balance in an experiment model of RA. Since 30% of patients do not respond to anti-TNF biotherapies, however, there is a need to develop alternative therapeutic approaches.

Objective

Strong association of other proinflammatory cytokines with the pathogenesis of RA prompted us to investigate which cytokine other than TNFα could be targeted for therapeutic benefit using RNA interference.

Methods

Two siRNA sequences were designed for IL-1β, IL-6 and IL-18 proinflammatory cytokines, and their efficacy and specificity were validated in vitro on J774.1 mouse macrophage cells, measuring both mRNA and protein levels following a lipopolysaccharide challenge. For in vivo administration, siRNAs were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA, and were injected intravenously in DBA/1 mice having collagen-induced arthritis. The clinical course of the disease was assessed by paw thickness 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 immunological balance was assessed using antitype II collagen assays. The distribution of siRNAs was evaluated by fluorometry in GFP transgenic mice over time after anti-GFP siRNA lipoplex injections.

Results

The designed siRNA sequences silenced 70–75% of the lipopolysaccharide-induced IL-1β, IL-6 and IL-18 mRNA expression in macrophages compared with a control siRNA. Each siRNA affected the targeted cytokine specifically, without modifying other proinflammatory cytokine mRNAs. In the collagen-induced arthritis model, weekly injections of siRNA lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling, and destruction of cartilage and bone, in both preventive and curative settings. The most striking therapeutic effect was observed when combining the three siRNAs targeting IL-1β/IL-6/IL-18 at once. Such tritherapy was associated with downregulation of both inflammatory and autoimmune components of the disease, and overall parameters were improved compared with the TNFα siRNA lipoplex-based treatment. The siRNA formulation was widely distributed, delivering the siRNA to several organs with a strong efficacy in the liver and spleen.

Conclusion

Tritherapy targeting IL-1β/IL-6/IL-18 seems highly effective to reduce all pathological features of RA including inflammation, joint destruction and Th1 response. These data show that cytokines other than TNFα can be targeted to improve symptoms of RA and reveal novel potential drug development targets. The systemic administration of anticytokine siRNA cocktails as a lipoplex could represent a novel and promising anti-inflammatory alternative therapy in RA.