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• W1992398204 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. 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. IntroductionThe use of synthetic double-stranded RNA oligonucleotides to trigger RNA interference (RNAi) and specifically to reduce the expression of targeted genes is a standard research method that is routinely used in vitro. The ability to manipulate gene expression levels can help to elucidate the functions of gene products and facilitate the observation of subsequent phenotypic changes in a controlled experimental fashion.1Hannon GJ RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3487) Google Scholar,2Agrawal N Dasaradhi PV Mohmmed A Malhotra P Bhatnagar RK Mukherjee SK RNA interference: biology, mechanism, and applications.Microbiol Mol Biol Rev. 2003; 67: 657-685Crossref PubMed Scopus (762) Google Scholar RNAi is also gaining acceptance as a research tool in vivo, and over a hundred publications already report use of this method in live animals.3Whelan J First clinical data on RNAi.Drug Discov Today. 2005; 10: 1014-1015Crossref PubMed Scopus (63) Google Scholar,4Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar In addition to elucidating gene function in vivo, it can be used for validating the potential significance of specific genes as targets for small-molecule drug development programs.5Sah DW Therapeutic potential of RNA interference for neurological disorders.Life Sci. 2006; 79: 1773-1780Crossref PubMed Scopus (75) Google Scholar Over 30 pharmaceutical and biotechnology companies are actively pursuing RNAi-based therapeutics, and several compounds are already in clinical trials. In spite of the rapid progress made in in vivo applications of RNAi, the field is still in its infancy; the methods in use are neither routine nor standardized. The use of small-interfering RNA (siRNA) in the central nervous system (CNS) may be more complex than its use in other organs such as the liver. In general, the greatest challenges to the in vivo use of RNAi relate to delivery (especially with target organ or cell type specificity) and to limiting the potential for undesired off-target effects (OTEs).6Kim JY Choung S Lee EJ Kim YJ Choi YC Immune activation by siRNA/liposome complexes in mice is sequence- independent: lack of a role for Toll-like receptor 3 signaling.Mol Cells. 2007; 24: 247-254PubMed Google ScholarsiRNAs are the natural products of processing longer double-stranded RNAs by the endoribonuclease Dicer.7Elbashir SM Harborth J Lendeckel W Yalcin A Weber K Tuschl T Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.Nature. 2001; 411: 494-498Crossref PubMed Scopus (8082) Google Scholar Most researchers today employ synthetic RNA duplexes that are 21 bases long as experimental tools to trigger RNAi. These molecules mimic the natural siRNAs produced by Dicer processing. In 2005, several groups demonstrated that the use of slightly longer synthetic RNAs, which are substrates for Dicer, can show higher potency than traditional 21-mer siRNAs.8Siolas D Lerner C Burchard J Ge W Linsley PS Paddison PJ et al.Synthetic shRNAs as potent RNAi triggers.Nat Biotechnol. 2005; 23: 227-231Crossref PubMed Scopus (367) Google Scholar,9Kim DH Behlke MA Rose SD Chang MS Choi S Rossi JJ Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.Nat Biotechnol. 2005; 23: 222-226Crossref PubMed Scopus (727) Google Scholar These longer RNAs are processed by Dicer into 21-mer siRNAs in a predictable manner,10Rose SD Kim DH Amarzguioui M Heidel JD Collingwood MA Davis ME et al.Functional polarity is introduced by Dicer processing of short substrate RNAs.Nucleic Acids Res. 2005; 33: 4140-4156Crossref PubMed Scopus (258) Google Scholar and the increased potency seen with this approach is thought to arise from the participation of Dicer in RNA-induced silencing complex formation (Figure 1). Typically, these reagents are synthetic RNA duplexes 27 bases in length and are referred to as Dicer-substrate siRNAs (DsiRNAs). An increasing number of reports have validated the performance of the Dicer-substrate reagents in vitro; however, given their relatively recent introduction, the number of reports showing in vivo activity are fewer in number.11Amarzguioui M Lundberg P Cantin E Hagstrom J Behlke MA Rossi JJ Rational design and in vitro and in vivo delivery of Dicer substrate siRNA.Nat Protoc. 2006; 1: 508-517Crossref PubMed Scopus (107) Google Scholar,12Kim M Shin D Kim SI Park M Inhibition of hepatitis C virus gene expression by small interfering RNAs using a tri-cistronic full-length viral replicon and a transient mouse model.Virus Res. 2006; 122: 1-10Crossref PubMed Scopus (37) Google Scholar Previous in vivo work demonstrated the use of DsiRNAs formulated with cationic lipids delivered by intraperitoneal injection to target macrophages, or the use of naked DsiRNAs delivered by hydrodynamic intravenous injection (tail vein) to target hepatocytes. In this study, we employ DsiRNAs to target specific cells within the CNS.The systemic delivery of siRNAs in vivo to peripheral organs and tumors has been demonstrated in a variety of systems (for recent reviews, see refs. 4Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar,13Aigner A Delivery systems for the direct application of siRNAs to induce RNA interference (RNAi) in vivo.J Biomed Biotechnol. 2006; 2006: 71659Crossref PubMed Scopus (113) Google Scholar). Because siRNAs do not cross the blood–brain barrier,14Pardridge WM Drug and gene delivery to the brain: the vascular route.Neuron. 2002; 36: 555-558Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar silencing of gene targets in the CNS requires either use of a blood–brain barrier-permeable carrier or direct injection into brain parenchyma or cerebrospinal fluid. Continuous intrathecal (IT) infusion of naked siRNA has been shown to be effective; however, very high doses are required.15Dorn G Patel S Wotherspoon G Hemmings-Mieszczak M Barclay J Natt FJ et al.siRNA relieves chronic neuropathic pain.Nucleic Acids Res. 2004; 32: e49Crossref PubMed Scopus (349) Google Scholar,16Altier C Dale CS Kisilevsky AE Chapman K Castiglioni AJ Matthews EA et al.Differential role of N-type calcium channel splice isoforms in pain.J Neurosci. 2007; 27: 6363-6373Crossref PubMed Scopus (123) Google Scholar Although toxic when injected intravenously, cationic lipids can be used in vivo to facilitate local delivery of nucleic acids. The cationic lipid i-Fect (Neuromics) was used by Luo and colleagues17Luo MC Zhang DQ Ma SW Huang YY Shuster SJ Porreca F et al.An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons.Mol Pain. 2005; 1: 29Crossref PubMed Scopus (149) Google Scholar to deliver siRNA through IT injection to target delta opioid receptors in the spinal cord. More recently, Kumar reported the use of siRNA delivered by direct intracerebral injection with i-Fect to protect mice from lethal viral encephalitis,18Kumar P Lee SK Shankar P Manjunath N A single siRNA suppresses fatal encephalitis induced by two different flaviviruses.PLoS Med. 2006; 3: e96Crossref PubMed Scopus (152) Google Scholar and Dong demonstrated the efficacy of IT infusion of siRNA formulated in i-Fect to silence the sodium channel Nav1.8 in rat dorsal root ganglia (DRG) in nociception.19Dong XW Goregoaker S Engler H Zhou X Mark L Crona J et al.Small interfering RNA-mediated selective knockdown of Na(V)1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats.Neuroscience. 2007; 146: 812-821Crossref PubMed Scopus (106) Google ScholarIn this study, we demonstrate the efficacy of 27-mer DsiRNA in reducing the expression of a specific G-protein coupled-receptor (GPCR) gene in rat spinal cord and DRG. Low doses of DsiRNA formulated in i-Fect, when administered by IT injection, induced a sustained reduction in the neurotensin receptor-2 (NTS2) GPCR mRNA and protein levels for 3–4 days. The reduction in NTS2 resulted in the expected behavioral changes in nociception. No apparent toxicity or nonspecific side effects were exhibited during the study period, and our results overall highlight the feasibility of using DsiRNA in pain research.ResultsNTS2 is a GPCR expressed in spinal and supraspinal structures involved in pain processing.22Dobner PR Neurotensin and pain modulation.Peptides. 2006; 27: 2405-2414Crossref PubMed Scopus (96) Google Scholar Stimulation of NTS2 results in analgesia. Evidences for its involvement in pain processing are based on the observed regulation of NTS2 expression in animal models of chronic pain and altered pain responses in knockout mice.23Maeno H Yamada K Santo-Yamada Y Aoki K Sun YJ Sato E et al.Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception.Brain Res. 2004; 998: 122-129Crossref PubMed Scopus (65) Google Scholar Additional direct proof is provided by the observed inhibition of neurotensin (NT)-induced analgesia by NTS2-specific antisense oligodeoxynucleotides after intracerebroventricular delivery,24Dubuc I Remande S Costentin J The partial agonist properties of levocabastine in neurotensin-induced analgesia.Eur J Pharmacol. 1999; 381: 9-12Crossref PubMed Scopus (43) Google Scholar and is further supported by the recent report of spinal analgesic activity of the selective-NTS2 agonist, JMV-431.21Sarret P Esdaile MJ Perron A Martinez J Stroh T Beaudet A Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors.J Neurosci. 2005; 25: 8188-8196Crossref PubMed Scopus (62) Google Scholar In view of the fact that we have already established a role for NTS2 in nociceptive modulation, this target was chosen for proof-of-concept demonstration of the role of DsiRNAs in silencing targets related to CNS disorders, such as pain states.In vitro validation of DsiRNAsWe synthesized and characterized six DsiRNAs specific for the rat NTS2 gene (NM_022695). These six DsiRNAs and an appropriate mismatch control (randomized RNA sequence not targeting any rat gene) were evaluated in vitro for their ability to specifically reduce NTS2 mRNA levels in an NTS2-stable Chinese hamster ovary cell line. The cells were treated with varying doses of DsiRNA, and the capacity of the latter to silence NTS2 was analyzed using quantitative reverse-transcription PCR. Three DsiRNAs tested, v1-1, v2-3, and v2-5, resulted in nearly complete inhibition of NTS2 expression at 10 nmol/l and remained effective at a very low dose (0.1 nmol/l) (Supplementary Figure S1). In addition to having high potency, duplex v1-1 has identical sequence between mouse and rat and potentially functions in both species (untested in mice).In order to confirm that dicing of DsiRNAs into the predicted 21-mer end-products does occur and that these are the active species loaded into RNA-induced silencing complex, 5′-RACE-PCR was performed with the v2-5 anti-NTS2 DsiRNA transfected into NTS2-expressing Chinese hamster ovary cells. Sequences of the transfected 27-mer DsiRNA and the predicted 21-mer siRNA product that should result from Dicer processing are shown in Supplementary Figure S2a. The 5′-RACE (rapid amplification and cloning of ends) procedure was performed using NTS2-specific primers, and five clones containing amplified NTS2 cDNA were recovered, all of them containing exactly the same sequence (Supplementary Figure S2b). The exact same internal cut site relative to the cloning linker was identified in all five clones. The alignment of the NTS2 mRNA sequence with the guide strands of the 27-mer and 21-mer species is shown in Supplementary Figure S2c, clearly identifying the cut site as lying between bases 10 and 11 relative to the 5′-end of the predicted 21-mer guide strand. We infer that Dicer processing of the 27-mer siRNA does occur in cells and that the resulting 21-mer species directs RNA-induced silencing complex cleavage of the NTS2 mRNA. On the basis of these earlier observations, two of these DsiRNAs (v1-1 and v2-5) were employed in subsequent in vivo studies to reduce the expression of NTS2 in rat spinal cord and DRG (Supplementary Table S1).Effective uptake of DsiRNAs by CNS tissuesBefore examining whether the administration of anti-NTS2 DsiRNAs altered the antinociceptive response mediated by NTS2 activation, we first evaluated whether they were able to reach targeted tissues after IT administration in a formulation with a cationic lipid. A control DsiRNA conjugated with Texas Red (2 administrations of either 1 μg or 5 μg with a 24-hour interval; n = 3) in the cationic lipid transfection agent i-Fect was delivered IT into the lumbar spinal cord of the rats. The rats were euthanized and the spinal cord and DRG were removed 24 hours after the last injection. Laser-scanning confocal microscopy revealed strong fluorescence signals in both lumbar DRG and spinal cord, thereby demonstrating effective uptake of DsiRNAs by IT infusion (Figure 2). Within the DRG, fluorescence staining was present in the cell bodies of different subpopulations of DRG neurons, with no apparent difference between the two tested groups (Figure 2a). The fact that not all primary sensory neurons showed fluorescence, and that the labeling intensity varied among the cells, makes it unlikely that unspecific staining or diffusion are responsible for the observed distribution. At a higher magnification, confocal images revealed that the fluorescence was confined to the cytoplasm of the cells and presented a diffuse cytoplasmic staining with added accumulation in what appeared to be endosome-like compartments (Figure 2b). Cellular uptake was also detected over the entire cross-section of the spinal cord, labeling both dorsal and ventral horn neurons (Figure 2c). Fluorescence was also detected in the neuronal cell processes, thereby indicating that DsiRNAs were taken up and transported within spinal nociceptive structures (Figure 2d).Figure 2Cellular uptake of Texas Red–tagged Dicer-substrate small-interfering RNA (DsiRNA) by spinal nociceptive structures. (a,b) Distribution of fluorescence in lumbar dorsal root ganglia at 24 hours after intrathecal injection of a control siRNA conjugated with Texas Red (1 μg administered twice with a 24-hour interval; n = 3). As seen by confocal microscopy, the staining is not uniformly distributed among the cells. Higher-magnification images also show that the fluorescent signal is detected in the form of small intracytoplasmic hot spots, sparing the nucleus. (c,d) Expression of Texas Red–tagged DsiRNA in a dorsal spinal cord section taken from an L5 segment. Fluorescence clusters are present in the cytoplasm of the cells. Note that the labeling is also detected in neuronal processes. Scale bar: 60 μm in a, 30 μm in b, 25 μm in c and 15 μm in d.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In vivo silencing of targeted gene expression by DsiRNA deliveryWe assessed the effects of IT administration of anti-NTS2 DsiRNAs (1 μg injected twice with a 24-hour interval) on NTS2 mRNA and protein expression levels using real-time quantitative reverse-transcription PCR and western blot analysis on DRG and spinal cord lysates. As compared to results from saline-treated rats and rats receiving the transfection reagent alone, a significant reduction of NTS2 mRNA was detected in the DRG and spinal dorsal horn at 24 hours after the last DsiRNA dose (Table 1). Use of the anti-NTS2 v2-5 DsiRNA resulted in an 86% decrease in NTS2 transcripts in the lumbar DRG as compared to vehicle controls, and 62% reduction in the spinal cord (n = 4). Similarly, the use of the v1-1 anti-NTS2 DsiRNA resulted in reduction in NTS2 expression by 52 and 70% in the DRG and the spinal cord, respectively (Table 1). Downregulation of NTS2 was confirmed at the protein level by western blot analysis (data not shown).Table 1Changes in NTS2 gene expression after in vivo DsiRNA deliveryExpression ratio NTS2/RPL23Salinei-FectDsíRNA v2-5DsíRNA v1-1Spinal cord1.27 ± 0.140.99 ± 0.160.49 ± 0.120.38 ± 0.13(22.2%)(61.6 %)**(70.4%)**DRG0.92 ± 0.110.61 ± 0.350.13 ± 0.050.44 ± 0.15(33.8 %)(85.5%)***(51.9%)*Abbreviations: DRG, dorsal root ganglia; DsiRNA, Dicer-substrate small-interfering RNA; NTS2, neurotensin receptor-2.NTS2 mRNA level was measured in spinal cord and DRG by quantitative reverse- transcription PCR 24 hours after a 2 day pretreatment with DsiRNA v1-1, DsiRNA v2-5, vehicle (i-Fect) or saline. The data are analyzed with the relative standard curve method (ΔΔCp) and normalized against internal control gene RPL23. Each value corresponds to the mean ± SEM of five biological samples done in triplicate and the knockdown percentage compared to the saline group. *P < 0.05, **P < 0.01, ***P < 0.001, compared to saline. Open table in a new tab DsiRNA as a tool for modulating painIn order to evaluate the efficacy of DsiRNA in silencing endogenous NTS2 receptor analgesic effects, we introduced duplexes targeting NTS2, v1-1, and v2-5 into the cerebrospinal fluid at the spinal level. Rats received an IT bolus administration of 1 μg DsiRNA, mismatch control, or vehicle once a day for 2 consecutive days, and the nociceptive response was measured each day starting at 24 hours and up to 4 days after the last DsiRNA injection, using the tail-flick test. In the vehicle-treated rats, repeated injections of the NTS2-selective agonist JMV-431 (90 μg/kg) elicited an antinociceptive effect over a 4-day period, characterized by an increase in tail-flick response latencies as compared to baseline values (producing up to 58% of the maximum possible effect; Figure 3). In contrast, IT-injected JMV-431 showed no influence on the nociceptive threshold in DsiRNA v1-1-treated animals; silencing of NTS2 reduced the analgesic effects of the JMV-431 by 93% (Figure 3a). The reversal of the JMV-431 response was no longer present on the fourth day after treatment, thereby suggesting a return of functional NTS2 receptors. When using RNAi as a research tool, a simple strategy to enhance confidence that findings are not biased by unsuspected OTEs is to require that more than one DsiRNA against a given target should produce the same biological results. We demonstrated that the treatment with a second anti-NTS2 DsiRNA v2-5 also blocked JMV-431-induced analgesia (inhibiting the JMV-431-induced antinociception by 99.5%; Figure 3b). As observed with DsiRNA v1-1, the functional inhibition of NTS2 by DsiRNA v2-5 was transient and reversible, because JMV-431-induced antinociception was not different from that of vehicle-treated rats within 3 days after the last DsiRNA injection (Figure 3b). Another strategy to ensure that misleading results do not occur because of crossreactivity with genes having sequence homology is to verify that closely related receptors are not functionally affected. DsiRNA-treated rats were given an IT administration of a NTS1-selective agonist, PD149163. As expected, the analgesic efficacy of the NTS1-selective drug was not affected by anti-NTS2 DsiRNA treatment (data not shown).Figure 3Knockdown of endogenous neurotensin receptor-2 (NTS2) receptors by Dicer-substrate small-interfering RNAs (DsiRNAs) blocked spinal antinociception of NTS2 agonist. The withdrawal latency was measured every day using the tail-flick test, starting at 24 hours after the last DsiRNA (1 μg/day for 2 consecutive days) or vehicle injection. (a) Pretreatment with DsiRNA v1-1 completely reverses JMV-431-induced antinociception for 72 hours, returning the latency of response to the baseline level of DsiRNA v1-1 pretreated rats injected with saline. (b) Pretreatment with the DsiRNA v2-5 also blocks the antinociceptive effect observed after administration of 90 μg/kg JMV-431 up to the third day, bringing the withdrawal latency back to the level of DsiRNA v2-5 pretreated animals injected with saline. Each data set represents mean values ± SEM from at least 6 animals. *P < 0.05, **P < 0.01, ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)An analysis of the behavioral experiments after IT injection of JMV-431 showed no statistical difference between vehicle-treated rats and rats receiving a nontargeting control DsiRNA (Figure 4). The mismatch control DsiRNA did not interfere with JMV-431 analgesic activity. Additionally, the baseline response latency in the absence of the NTS2 agonist was similar in vehicle-treated rats, in rats receiving the control DsiRNA, as well as in DsiRNA v1-1- and DsiRNA v2-5-treated rats (Figure 4). That is, in vivo silencing of NTS2 by the NTS2-specific DsiRNAs did not modify basal thermal nociception; this finding is consistent with previous reports.23Maeno H Yamada K Santo-Yamada Y Aoki K Sun YJ Sato E et al.Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception.Brain Res. 2004; 998: 122-129Crossref PubMed Scopus (65) Google Scholar,24Dubuc I Remande S Costentin J The partial agonist properties of levocabastine in neurotensin-induced analgesia.Eur J Pharmacol. 1999; 381: 9-12Crossref PubMed Scopus (43) Google Scholar No side effects, such as locomotion impairment, weight loss, discomfort, or body temperature modification were observed. The rats behaved normally during the entire period of treatment with either DsiRNAs or NT receptor agonists. Although the DsiRNAs were delivered IT, there were also no signs of spinal cord or DRG inflammation at the time of dissection and histological examination. Taken together, these results demonstrate that the use of DsiRNA is a powerful tool for the modulation of nociceptive pathways, and is safe, at least for short-term administration.Figure 4Basal nociceptive responses to thermal stimuli. Intrathecal injection of neurotensin receptor-2 Dicer-substrate small-interfering RNA (DsiRNA) v1-1 or DsiRNA v2-5 does not modify the tail withdrawal latency in the acute pain paradigm as compared to vehicle (i-Fect) or mismatch DsiRNA injection. Additionally, mismatch DsiRNA does not affect JMV-431-induced analgesia. Each data set represents the mean values ± SEM from at least 15 animals. **P < 0.01, ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionThe use of RNAi is an emerging technology in gene therapy and greatly broadens the set of molecular tools available for basic research in vivo. Although this is already considered to be a robust technology, improvements that increase efficiency at low doses are still desirable, particularly for in vivo applications. This study demonstrates the potency of 27-mer DsiRNA in achieving an efficient knockdown of a GPCR in the CNS. Evolving pathologies, such as pain, often become drug-resistant in a large subset of the population, and there is a need for greater understanding of nociceptive mechanisms and for novel therapeutic approaches to support existing treatments. The DsiRNA method demonstrated here offers a potential new avenue to address this problem.RNAi-based therapies present certain advantages in comparison with traditional pharmacological approaches. Nucleic acid hybridization is very specific; theoretically, any desired gene could be uniquely targeted by an antisense or RNAi oligonucleotide of a complementary sequence.25Aagaard L Rossi JJ RNAi therapeutics: principles, prospects and challenges.Adv Drug Deliv Rev. 2007; 59: 75-86Crossref PubMed Scopus (715) Google Scholar That is, any gene can be identified and uniquely silenced, even the “nondruggable” targets that are inaccessible to the small-molecules pharmacopoeia.5Sah DW Therapeutic potential of RNA interference for neurological disorders.Life Sci. 2006; 79: 1773-1780Crossref PubMed Scopus (75) Google Scholar Over 30 pharmaceutical and biotechnology companies are conducting research in the area of RNAi therapeutics.4Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar Four “siRNA therapy” open studies, ranging from preclinical to phase III, are currently identified in the National Institutes of Health/Food and Drug Administration-supported registry of federally and privately supported clinical trials conducted in the United States and around the world.26ClinicalTrials.gov Information on Clinical Trials and Human Research Studies <http://www.ClinicalTrials.gov>Google Scholar Much of this work is focused on peripheral pathologies such as hepatitis C, respiratory viruses, macular degeneration, and human immunodeficiency virus to name only a few.4Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google ScholarThere currently are no active or pending clinical trials involving RNAi therapy for disorders of the CNS, although work in this area is ongoing. One of the problems underlying the delay in applying RNAi to CNS diseases relates to poor siRNA delivery by bolus injection methods and the side effects from the microsurgical implantation of sustained delivery devices. A single dose is seldom useful in inducing sustained silencing in the brain with stereotaxic injections or in the spine with IT injections. A handful of studies focusing on locomotion, pain, and learning disorders showed that central delivery of siRNAs induced functional silencing in the brain and in the spine.5Sah DW Therapeutic potential of RNA interference for neurological disorders.Life Sci. 2006; 79: 1773-1780Crossref PubMed Scopus (75) Google Scholar Although successful, they reported a number of limitations that would hamper progress toward an effective and sustained knockdown using low siRNA concentrations;27Hoyer D Thakker DR Natt F Maier R Huesken D Muller M et al.Global down-regulation of gene expression in the brain using RNA interference, with emphasis on monoamine transporters and GPCRs: implications for target characterization in psychiatric and neurological disorders.J Recept Signal Transduct Res. 2006; 26: 527-547Crossref PubMed Scopus (17) Google Scholar e.g., some of these studies needed to employ dosing as high as 400 μg/day of continuous" @default.
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• W1992398204 title "Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research" @default.
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• W1992398204 doi "https://doi.org/10.1038/mt.2008.98" @default.
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