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Agenda Topics:
Target Discovery and Validation
Transfection
siRNA Therapeutics
in vivo RNAi-recent in vivo RNAi Pubs
Bioinformatics of small RNAs
siRNA library screens
microRNAs in
Disease Biology:

Stem Cell Biology

Diagnostics

Virology

Biogenesis

Development

RNAi Researchers Galvanized by Advances

Technology's Viability in Drug Development Is Finally Established
Author: Elizabeth Lipp
Publication: Genetic Engineering & biotechnology News
Publisher: Mary Ann Liebert, Inc. publishers
Date: Jun 1, 2008
Copyright © 2008 GEN Publishing

Article Link: http://www.genengnews.com/articles/chitem.aspx?aid=2493

Notable Quotables:

“Long dsRNAs have been employed for many years as a means to modulate gene expression in plants, yeast, and C. elegans,” noted Mark Behlke, M.D., Ph.D., svp of molecular genetics and CSO at Integrated DNA Technologies (IDT; www.idtdna.com).
“Similar attempts in higher organisms failed due to interferon activation, however we now know that short RNA duplexes can be safely used in mammalian systems both in vitro and in vivo. The technology has rapidly matured, thanks in large part to all that was learned over the past 20 years using antisense oligonucleotides. RNAi is now routinely employed in vivo as an experimental tool and numerous groups are vigorously pursing the use of RNAi compounds as therapeutics. Several siRNA drugs are already in clinical trials and more are in preclinical development.”

Hi Thru Put Delivery of siRNA/DsiRNA

NEW PRODUCT

i-Fect ™ Hi Put 96-New

Learn about transfection products @ neuromics

Central Delivery of DsiRNA

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); doi:10.1038/mt.2008.98.

Images: Cellular 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. Courtesy of Dr. Nicolas Beudeat. Published in Molecular Therapy (2008); doi:10.1038/mt.2008.98

Use of Dicer Substrate siRNAs

Dicer-substrate siRNAs (DsiRNAs) have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration. “IDT got into RNAi research in collaboration with John Rossi at The City of Hope and the Beckman Research Institute five years ago,” explained Dr. Behlke. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RISC and serve as a sequence-specific guide to target degradation of complementary mRNA species.
Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3´-overhangs on the termini, reported Dr. Behlke. These duplexes are transfected into cells lines, directly mimicking the products made by dicer in vivo. Most siRNA sequences can be administered to cultured cells or to animals without eliciting an interferon response.

“We observed,” added Dr. Behlke, “that the use of slightly longer sequences that were substrates for dicer showed improved potency, which we theorize relates to participation of dicer in RISC loading. We are now focusing on the use of these compounds in vivo.”

IDT recently completed a collaborative study with the laboratory of professor Phillipe Sarret at the Université de Sherbrooke in Quebec. The collaboration studied the use of DsiRNA to knockdown the GPCR NTS2 (neurotensin type 2 receptor) in rat spinal cord and dorsal root ganglia. The RNA duplexes were administered by intrathecal injection in a cationic lipid slurry. Stimulation of NTS2 with a chemical agonist resulted in analgesia. Pain responses were monitored in treated animals by dipping their tails in hot water with and without the chemical agonist.

“The anti-NTS2 DsiRNA treated animals showed a marked difference of response to the test stimulus,” said Dr. Behlke. “We recorded differences of up to five seconds, which is quite a long time for a rat to sit with its tail in hot water. While interesting, this work mainly represents a pilot study to demonstrate the feasibility of using DsiRNA to study pain pathways in rats. We were amazed at the low dose it takes to get knockdown—we used 1 mcg/200 g rat, which is only a 0.005 mg/kg dose.” Modulating CNS disease and affecting brain processes is clearly possible, but better methods of delivery are going to be needed to move this approach from a research tool into the clinic, noted Dr. Behlke.

Ret Knockdown Using Small Interference RNA

Nearly 100% Transfection Efficiency Reported in vitro with i-Fect ™.

Images: Ret receptor knockdown using small interference RNA (siRNA) in podocytes. (A) Transfection efficiency: mouse podocytes were transfected with 100 nM concentrations of Ret siRNA or vehicle alone. (a) When podocytes were exposed to i-Fect alone, there was no toxicity. (a and b) A transfection efficiency of nearly 100% was achieved with 100 nM concentration of Ret siRNA (b). Co-transfection with a fluorescently tagged control siRNA was used to determine the transfection efficiency, and fluorescence microscopy revealed a perinuclear localization of the tagged RNA (b, arrowhead). (B) Western blot analysis of Ret after transfection: Ret immunoblotting (top) of WCL 2, 3, or 4 d after transfection revealed that Ret was downregulated within 2 d after transfection with 100 nM Ret siRNA. Transfection of control siRNA at day 4 served as a negative control, and the maximal knockdown of Ret was observed 4 d after transfection. GAPDH immunoblotting confirmed equal protein loading (bottom). doi:10.1681/ASN.2005080835. (full text publication)

Methods: Ret small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled functionally validated Ret siRNA (SMARTpool mouse RET siRNA; Dharmacon, Lafayette, CO). HSMP cells that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above and transfected using the i-Fect siRNA
transfection reagent (Neuromics, Northfield, MN). For determination
of the transfection efficiency, a Texas Red–labeled siRNA (siGLO RISCFree
siRNA; Dharmacon) was co-transfected with Ret siRNA and visualized
using fluorescence microscopy. For control siRNA samples,
identical conditions were used with the substitution of siGLO-RISCFree
siRNA for Ret siRNA. For determination of the efficiency of Ret
knockdown, Western analysis for Ret was performed on WCL from
cells 24 to 96 h after the transfection. Several concentrations of Ret
siRNA (40, 60, and 100 nM) were tested to determine optimal knockdown
conditions. For apoptosis assays, podocytes were exposed to
UV-C or PA (40 g/ml) on days 3 to 4 after transfection of Ret siRNA
or control siRNA (100 nM). Apoptosis was measured by counting
podocytes with Hoechst-positive pyknotic nuclei 3 h after UV and 5 h
after exposure to PA.

RNAi therapeutics: a potential new class of pharmaceutical drugs

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

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.