Electro-transfer of small interfering RNA ameliorated arthritis in rats

doi:10.1016/j.bbrc.2005.08.198

Biochemical and Biophysical Research Communications

Volume 336, Issue 3, 28 October 2005, Pages 903-908

https://doi.org/10.1016/j.bbrc.2005.08.198 Get rights and content

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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The goal of this feasibility study was to examine whether sonoporation assisted transduction of siRNA could be used to ameliorate arthritis locally. If successful, such approach could provide an alternative treatment for the patients that have or gradually develop adverse response to chemical drugs. Tumor necrosis factor alpha (TNF-α) produced by synovial fibroblasts has an important role in the pathology of rheumatoid arthritis, inducing inflammation and bone destruction. In this study, we injected a mixture of microbubbles and siRNA targeting TNF-α (siTNF) into the articular joints of rats, and transduced siTNF into synovial tissue by exposure to a collimated ultrasound beam, applied through a probe 6 mm in diameter with an input frequency of 3.0 MHz, an output intensity of 2.0 W/cm² (spatial average temporary peak; SATP), a pulse duty ratio of 50%, and a duration of 1 min. Sonoporation increased skin temperature from 26.8 °C to 27.3 °C, but there were no adverse effect such as burns. The mean level of TNF-α expression in siTNF-treated knee joints was 55% of those in controls. Delivery of siTNF into the knee joints every 3 days (i.e., 7, 10, 13, and 16 days after immunization) by in vivo sonoporation significantly reduced paw swelling on days 20–23 after immunization. Radiographic scores in the siTNF group were 56% of those in the CIA group and 61% of those in the siNeg group. Histological examination showed that the number of TNF-α positive cells was significantly lower in areas of pannus invasion into the ankle joints of siTNF- than of siNeg-treated rats. These results indicate that transduction of siTNF into articular synovium using sonoporation may be an effective local therapy for arthritis.
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The aim of this study was to investigate potentialities of poly(dl-lactide–co-glycolide) (PLGA) microspheres for the delivery of small interfering RNAs (siRNAs) against tumor necrosis factor α (TNF-α) to achieve prolonged and efficient inhibition of TNF-α for the treatment of rheumatoid arthritis (RA). PLGA microspheres were prepared by a modified multiple emulsion–solvent evaporation method. The formulations were characterized in terms of morphology, mean diameter and siRNAs distribution, encapsulation efficiency, and in vitro release kinetics. The efficiency of this system was then evaluated both in vitro and in vivo using the murine monocytic cell line J774 and a pre-clinical model of RA, respectively. siRNA-encapsulating PLGA microspheres were characterized by a high encapsulation efficiency and a slow and prolonged anti-TNF-α siRNAs. Our results provide evidence that, upon intra-articular administration, PLGA microspheres slowly releasing siRNAs effectively inhibited the expression of TNF-α in arthritic joints. Our system might represent an alternative strategy for the design of novel anti-rheumatic therapies based on the use of RNA interference in RA.
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This was demonstrated by a significant improvement in arthritis symptoms such as joint swelling, expression of proinflammatory cytokines, and articular histological degradation. The efficiency of both approaches showed that it is advisable to inhibit gene expression at an early stage of protein synthesis in articular cells.8,19 Furthermore, we observed that the effects of the TFO injection were much greater than with the same amount of siRNA (10 µg).
Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including arthritis. Neutralization of this cytokine by anti-TNF-α antibodies has shown its efficacy in rheumatoid arthritis (RA) and is now widely used. Nevertheless, some patients currently treated with anti-TNF-α remain refractory or become nonresponder to these treatments. In this context, there is a need for new or complementary therapeutic strategies. In this study, we investigated in vitro and in vivo anti-inflammatory potentialities of an anti-TNF-α triplex-forming oligonucleotide (TFO), as judged from effects on two rat arthritis models. The inhibitory activity of this TFO on articular cells (synoviocytes and chondrocytes) was verified and compared to that of small interfering RNA (siRNA) in vitro. 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. Furthermore, the TFO efficiently blocked synovitis and cartilage and bone destruction in the joints. 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.

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Electro-transfer of small interfering RNA ameliorated arthritis in rats

Abstract

Section snippets

Materials and methods

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

Discussion

Acknowledgments

J. Mol. Biol.

Lancet

Biochem. Biophys. Res. Commun.

FEBS Lett.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Nature

RNA interference

Nature

Gene silencing as an adaptive defence against viruses

Nature

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Nature

Efficient delivery of siRNA for inhibition of gene expression in postnatal mice

Nat. Genet.

RNA interference in adult mice

Nature

Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA

J. Gene Med.

RNA interference targeting Fas protects mice from fulminant hepatitis

Nat. Med.

Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene

Nat. Med.

In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury

J. Leukoc. Biol.

RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis

Mol. Vis.

RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia

Nat. Med.

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference

Proc. Natl. Acad. Sci. USA

Exploring RNA interference as a therapeutic strategy for renal disease

Gene Ther.