Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research

Abstract

RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3-4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs.

Chemical Modification of siRNAs forIn VivoUse

“…Similarly, 21-mer siRNAs are currently the lead compounds for a number of clinical and preclinical RNAi drug development programs. Alternative designs are also in use for both research and drug development, including blunt 19-mers (Czauderna et al, 2003;Allerson et al, 2005;Prakash et al, 2005;Kraynack and Baker, 2006), blunt 25-mers (Chen et al, 2005), blunt 27-mers , and asymmetric 25/27-mers or 27/29-mers Dore-Savard et al, 2008;Nishina et al, 2008). Some of these compounds directly load into RISC whereas others are substrates for Dicer and are processed into shorter species before RISC loading.…”

RNAi: a potential new class of therapeutic for human genetic disease

“…For example, RNAi has been used to reduce neuropathic pain, prevent neuronal death induced by brain ischemia, minimize the damage caused by prion proteins, and induce apoptosis or increase chemotherapy susceptibility in brain tumors (Gondi et al 2004;Zhang et al 2004;Pfeifer et al 2006;Dore-Savard et al 2008;White et al 2008;Sun et al 2009). …”

Spinal NTS2 receptor activation reverses signs of neuropathic pain

“…Indeed, central delivery of stable NT analogs produces dose-dependent antinociception in response to different experimetital pain modalities, including thermal, mechanical, and chemical stimuli (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Accordingly, inhibition of NTSl or NTS2 function by pharmacological NT receptor antagonists, antisense technologies, or generation of knockout animals supports the involvement of these receptors in the regulation of pain processing (19,25,(28)(29)(30)(31)(32)(33)(34). In light of the fact that neuropathic pain conditions are only partially responsive to opioid therapy, it is also important to point out that NT exerts its analgesic effects at least in part through opioid-independent mechanisms (35,36).…”