Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo

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Abstract

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.

Section snippets

Materials and methods

Oligonucleotides and chemicals. Purified 19mer siRNA duplexes were purchased from IBA GmbH (Göttingen, Germany). Sequences of the siRNAs used in the present study are summarized in Table 1. The siRNAs were dissolved and annealed in DEPC-treated water for in vivo administrations. BCTC (N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide; Grünenthal GmbH, Germany) was dissolved in 10% DMSO, 5% Cremophor EL, in 5% glucose solution and administered in a volume of

Silencing of TRPV1 in cell culture

In a previous study, we screened six siRNAs to obtain an efficient inhibitor of TRPV1 expression [13]. Here, we performed a more detailed analysis of the concentration dependency of the two most efficient siRNAs in co-transfection experiments with a plasmid encoding the cDNA of a TRPV1-GFP fusion protein. As can be seen in Fig. 1, we observed the expected concentration-dependent silencing of target gene expression. Both siRNAs tested almost completely inhibited expression of TRPV1-GFP even at

Discussion

A crucial point in the development of new analgesic drugs is the validation of new targets for low molecular weight compounds. Here, we evaluate the potential of intrathecally injected siRNAs as tools for rapid functional investigation of pain targets. We chose the vanilloid receptor, TRPV1, as a target, which is considered a central molecular integrator of nociceptive signaling [6].

In vivo delivery of siRNAs into the central nervous system is complicated by the fact that oligonucleotides do

Acknowledgments

The authors thank Birgit Bieber, Johanna Korioth, Simone Pfennings, Elke Schumacher, Elke Frank-Rodenberg, and Denise Werk for excellent technical assistance. Financial support by the German Ministry for Research and Technology (BMBF, Grant No. 01GG9819/0), the RiNA network for RNA technologies, and the Fonds der Chemischen Industrie is gratefully acknowledged.

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    These authors contributed equally to this work.

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