Electro-transfer of small interfering RNA ameliorated arthritis in rats

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Abstract

RNA interference provides the powerful means of sequence-specific gene silencing. Particularly, small interfering RNA (siRNA) duplexes may be potentially useful for therapeutic molecular targeting of human diseases, although novel delivery systems should be devised to achieve efficient and organ-specific transduction of siRNA. In the present study, we demonstrated that electro-transfer of a siRNA–polyamine complex made efficient and specific gene knockdown possible in the articular synovium. Targeted suppression of the tumor necrosis factor-α gene through this procedure significantly ameliorated collagen-induced arthritis in rats. Our results suggest the potential feasibility of therapeutic intervention with RNA medicines for treatment of rheumatoid and other locomotor diseases.

Section snippets

Materials and methods

siRNA duplexes. siRNA duplexes targeting rat TNF-α gene were synthesized (A: 5′-GCCCGUAGCCCACGUCGUAd(TT)-3′ and 5′-UACGACGUGGGCUACGGGCd(TT)-3′; B: 5′-UGGGCUCCCUCUCAUCAGUd(TT)-3′ and 5′-ACUGAUGAGAGGGAGCCCAd(TT)-3′; C: 5′-GGAGGAGAAGUUCCCAAAUd(TT)-3′, and 5′-AUUUGGGAACUUCUCCUCCd(TT)-3′; and D: 5′-AGACAACCAACUGGUGGUAd(TT), and 5′-UACCACCAGUUGGUUGUCUd(TT)-3′). Two nucleotide mismatches were introduced into the TNF-α-siRNA-A to generate the mismatched TNF-α-siRNA (5′-GCCCGUAGAACACGUCGUAd(TT)-3′ and

Electro-transfer of siRNA into the synovium of the rat knee joint

GAPDH-specific siRNA duplex was labeled with 6-carboxyfluorescein (FAM) and injected into the left knee joint of DA rats, to which electric field was applied subsequently at the joint. Twenty-four hours later, the labeled siRNA was present at the intra-articular synovial tissue as revealed by fluorescence stereomicroscopic observation (data not shown). The siRNA was more effectively transduced when it was conjugated with a polyamine (siPORT Amine) before electro-transfer (Figs. 1A–D). The

Discussion

Therapeutic application of RNAi has been performed against a variety of disorders including hepatic [8], respiratory [10], [11], ocular [12], neuronal [13], [14], renal [15], and malignant [16], [17], [18], [19] diseases. To obtain therapeutic gene silencing in the liver, synthetic siRNA was intravenously administered to the animals [8], while RNAi in other organs was induced by siRNA [11], [12], [14], [16], [17], [19], [20] or a siRNA-expression plasmid [10], [18] that were administered into

Acknowledgments

We thank Dr. M. Okabe for supplying the EGFP transgenic rat. This study was supported by a Grant-in-Aid for Scientific Research (No. 15790799) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and JSPS Fujita Memorial Fund for Medical Research.

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