Comprehensive and Cogent overview on delivery methods:
David Bumcrot1, Muthiah Manoharan1, Victor Koteliansky1 and Dinah W Y Sah1. RNAi therapeutics: a potential new class of pharmaceutical drugs
Nature Chemical Biology 2, 711-719 (2006) doi:10.1038/nchembio839
Monday, November 26, 2007
Wednesday, November 7, 2007
i-Fect and Posters @ SfN
Program#/Poster#:
72.13/EE20
Title:
RNAi of neuropeptide Y for neuropathic pain
Location:
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Saturday, Nov 03, 2007, 1:00 PM - 2:00 PM
Authors:
*M.-C. LUO1, D. ZHANG1, E.-T. ZHANG1, Q. CHEN1, P. GE2, D. SAH2, T. VANDERAH1, F. PORRECA1, J. LAI1; 1Dept Pharmacol, Univ. Arizona Hlth. Sci. Ctr., Tucson, AZ; 2Alnylam Pharmaceuticals, Inc., Boston, MA
Neuropeptide Y (NPY) is upregulated after L5/L6 spinal nerve ligation (SNL) injury in large diameter dorsal root ganglion (DRG) neurons that project, via the ipsilateral dorsal column, to the brain stem gracile nucleus. Action of NPY in the gracile nucleus promotes hypersensitivity to innocuous touch, which mimics neuropathic pain in human (Ossipov et al., 2002). We hypothesize that a knock down of NPY in the injured DRG by small interfering RNA (siRNA) blocks the nerve injury induced tactile hypersensitivity.A number of synthetic siRNA that target preproNPY were screened by transfecting the cell line, F-11, that expresses endogenous NPY. A maximal knock down of 89% of preproNPY mRNA and 47% of the peptide was observed in vitro. The knock down of both mRNA and peptide lasted at least 72 hr following a single transfection. Sequence specificity of the siRNA-mediated knock down of NPY is confirmed by mismatch RNA control.A siRNA for preproNPY was delivered intrathecally to the lumbar spinal cord once daily at 2 μ g (with the vehicle i-Fect) in rats beginning one day prior to SNL for 7 days. SiRNA, but not mismatch RNA treatment, significantly attenuated tactile hypersensitivity in the injured paw. A moderate attenuation of NPY expression was confirmed by NPY-immunoreactivity in lumbar spinal cord and DRG in siRNA treated rats. Tactile hypersensitivity returned after cessation of siRNA treatment. siRNA treatment initiated after the tactile hypersensitivity was established was ineffective in reversing the abnormal pain behavior. Thus, early but not delayed intervention of NPY expression in the injured nerve significantly attenuated nerve injury induced tactile hypersensitivity, which is likely due to limited efficacy of the siRNA against a highly abundant gene target. Effect of siRNA may be further optimized in vivo by chemical stabilization and delivery. This study is supported by NIH grant R01NS046785.
Disclosures:
M. Luo , None; D. Zhang, None; E. Zhang, None; Q. Chen, None; P. Ge, None; D. Sah, None; T. Vanderah, None; F. Porreca, None; J. Lai, None.
Support:
NIH grant R01NS046785
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.
Program#/Poster#:
509.6/PP9
Title:
Small interfering RNA-mediated selective knockdown of NTS2 receptors reverses neurotensin-induced analgesia in rats
Location:
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Monday, Nov 05, 2007, 2:00 PM - 3:00 PM
Authors:
*L. DORE-SAVARD1,2, G. ROUSSY1, M.-A. DANSEREAU1, K. BELLEVILLE1, N. BEAUDET1, M. BEHLKE2, P. SARRET1; 1Physiology and Biophysics, Univ. Sherbrooke, Sherbrooke, PQ, Canada; 2Integrated DNA Technologies Inc., Coralville, IA
We have previously shown that NTS2 receptors play an important role in the regulation of nociceptive functions at the spinal level. Indeed, intrathecal (i.t.) administration of NTS2-selective agonist, levocabastine and JMV-431, induced a dose-dependent antinociceptive responses in the tail-flick test. Recent discoveries revealed that the delivery of small interfering RNA (siRNA) in vivo resulted in the potent, long-lasting, post-transcriptional silencing of specific genes. Thus, we investigated the effect of i.t. injection of siRNA targeting NTS2 receptors for the modulation of pain. Using Real-time PCR analysis, we first identified several siRNA capable of a high-selective attenuation of NTS2 message in rNTS2 stably transfected CHO cells. Dicer-substrate siRNA (DsiRNA), which have been shown to have increased potency in vitro compared to 21-mers, were therefore administered i.t. at the lumbar spinal cord level on days 0 and 1 at a dose of 1 µg formulated in the cationic lipid i-Fect transfection agent. Twenty-four hours after the last dose of DsiRNA, NTS2 protein levels were markedly reduced when examined by Western blot in dorsal root ganglia (DRG, 43.4%) and spinal cord (27.4%), compared to rats receiving control DsiRNA. Rats were then tested for antinociception by the NTS2-selective agonist, JMV-431 in the tail-flick test. Pretreatment with the DsiRNA targeting NTS2, but not the mismatch RNA or vehicle alone reduced by 93.3% the analgesic effects of JMV-431. The functional inhibition of NTS2 by DsiRNA was progressively reversed within 4 days after the last RNA injection. Texas Red-labeled DsiRNA were clearly detected in the cytoplasm of both lumbar DRG and spinal cord neurons, indicating that DsiRNA were taken up and transported within spinal nociceptive structures. Taken together, these results demonstrate that silencing of NTS2 receptors using a DsiRNA approach abolishes NT-induced antinociception and further support a role for NTS2 in the management of acute pain.
Disclosures:
L. Dore-Savard, None; G. Roussy, None; M. Dansereau, None; K. Belleville, None; N. Beaudet, None; M. Behlke, None; P. Sarret, None.
Support:
CIHR, FRSQ and CRS
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.2007 Copyright by the Society for Neuroscience all rights reserved. Permission to republish any abstract or part of any abstract in any form must be obtained in writing by SfN office prior to publication.
72.13/EE20
Title:
RNAi of neuropeptide Y for neuropathic pain
Location:
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Saturday, Nov 03, 2007, 1:00 PM - 2:00 PM
Authors:
*M.-C. LUO1, D. ZHANG1, E.-T. ZHANG1, Q. CHEN1, P. GE2, D. SAH2, T. VANDERAH1, F. PORRECA1, J. LAI1; 1Dept Pharmacol, Univ. Arizona Hlth. Sci. Ctr., Tucson, AZ; 2Alnylam Pharmaceuticals, Inc., Boston, MA
Neuropeptide Y (NPY) is upregulated after L5/L6 spinal nerve ligation (SNL) injury in large diameter dorsal root ganglion (DRG) neurons that project, via the ipsilateral dorsal column, to the brain stem gracile nucleus. Action of NPY in the gracile nucleus promotes hypersensitivity to innocuous touch, which mimics neuropathic pain in human (Ossipov et al., 2002). We hypothesize that a knock down of NPY in the injured DRG by small interfering RNA (siRNA) blocks the nerve injury induced tactile hypersensitivity.A number of synthetic siRNA that target preproNPY were screened by transfecting the cell line, F-11, that expresses endogenous NPY. A maximal knock down of 89% of preproNPY mRNA and 47% of the peptide was observed in vitro. The knock down of both mRNA and peptide lasted at least 72 hr following a single transfection. Sequence specificity of the siRNA-mediated knock down of NPY is confirmed by mismatch RNA control.A siRNA for preproNPY was delivered intrathecally to the lumbar spinal cord once daily at 2 μ g (with the vehicle i-Fect) in rats beginning one day prior to SNL for 7 days. SiRNA, but not mismatch RNA treatment, significantly attenuated tactile hypersensitivity in the injured paw. A moderate attenuation of NPY expression was confirmed by NPY-immunoreactivity in lumbar spinal cord and DRG in siRNA treated rats. Tactile hypersensitivity returned after cessation of siRNA treatment. siRNA treatment initiated after the tactile hypersensitivity was established was ineffective in reversing the abnormal pain behavior. Thus, early but not delayed intervention of NPY expression in the injured nerve significantly attenuated nerve injury induced tactile hypersensitivity, which is likely due to limited efficacy of the siRNA against a highly abundant gene target. Effect of siRNA may be further optimized in vivo by chemical stabilization and delivery. This study is supported by NIH grant R01NS046785.
Disclosures:
M. Luo , None; D. Zhang, None; E. Zhang, None; Q. Chen, None; P. Ge, None; D. Sah, None; T. Vanderah, None; F. Porreca, None; J. Lai, None.
Support:
NIH grant R01NS046785
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.
Program#/Poster#:
509.6/PP9
Title:
Small interfering RNA-mediated selective knockdown of NTS2 receptors reverses neurotensin-induced analgesia in rats
Location:
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Monday, Nov 05, 2007, 2:00 PM - 3:00 PM
Authors:
*L. DORE-SAVARD1,2, G. ROUSSY1, M.-A. DANSEREAU1, K. BELLEVILLE1, N. BEAUDET1, M. BEHLKE2, P. SARRET1; 1Physiology and Biophysics, Univ. Sherbrooke, Sherbrooke, PQ, Canada; 2Integrated DNA Technologies Inc., Coralville, IA
We have previously shown that NTS2 receptors play an important role in the regulation of nociceptive functions at the spinal level. Indeed, intrathecal (i.t.) administration of NTS2-selective agonist, levocabastine and JMV-431, induced a dose-dependent antinociceptive responses in the tail-flick test. Recent discoveries revealed that the delivery of small interfering RNA (siRNA) in vivo resulted in the potent, long-lasting, post-transcriptional silencing of specific genes. Thus, we investigated the effect of i.t. injection of siRNA targeting NTS2 receptors for the modulation of pain. Using Real-time PCR analysis, we first identified several siRNA capable of a high-selective attenuation of NTS2 message in rNTS2 stably transfected CHO cells. Dicer-substrate siRNA (DsiRNA), which have been shown to have increased potency in vitro compared to 21-mers, were therefore administered i.t. at the lumbar spinal cord level on days 0 and 1 at a dose of 1 µg formulated in the cationic lipid i-Fect transfection agent. Twenty-four hours after the last dose of DsiRNA, NTS2 protein levels were markedly reduced when examined by Western blot in dorsal root ganglia (DRG, 43.4%) and spinal cord (27.4%), compared to rats receiving control DsiRNA. Rats were then tested for antinociception by the NTS2-selective agonist, JMV-431 in the tail-flick test. Pretreatment with the DsiRNA targeting NTS2, but not the mismatch RNA or vehicle alone reduced by 93.3% the analgesic effects of JMV-431. The functional inhibition of NTS2 by DsiRNA was progressively reversed within 4 days after the last RNA injection. Texas Red-labeled DsiRNA were clearly detected in the cytoplasm of both lumbar DRG and spinal cord neurons, indicating that DsiRNA were taken up and transported within spinal nociceptive structures. Taken together, these results demonstrate that silencing of NTS2 receptors using a DsiRNA approach abolishes NT-induced antinociception and further support a role for NTS2 in the management of acute pain.
Disclosures:
L. Dore-Savard, None; G. Roussy, None; M. Dansereau, None; K. Belleville, None; N. Beaudet, None; M. Behlke, None; P. Sarret, None.
Support:
CIHR, FRSQ and CRS
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.2007 Copyright by the Society for Neuroscience all rights reserved. Permission to republish any abstract or part of any abstract in any form must be obtained in writing by SfN office prior to publication.
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