Monday, November 26, 2007

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

Wednesday, November 7, 2007

i-Fect and Posters @ SfN

RNAi of neuropeptide Y for neuropathic pain
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Saturday, Nov 03, 2007, 1:00 PM - 2:00 PM
*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.
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.
NIH grant R01NS046785
[Authors]. [Abstract Title]. Program No. XXX.XX. 2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Online.

Small interfering RNA-mediated selective knockdown of NTS2 receptors reverses neurotensin-induced analgesia in rats
San Diego Convention Center: Halls B-H
Presentation Start/End Time:
Monday, Nov 05, 2007, 2:00 PM - 3:00 PM
*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.
L. Dore-Savard, None; G. Roussy, None; M. Dansereau, None; K. Belleville, None; N. Beaudet, None; M. Behlke, None; P. Sarret, None.
[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.

Tuesday, October 30, 2007

More on Delivering siRNA in vivo

We are excited about this upcoming presentation at Society for Neuroscience comference presesentation. Neuromics' i-Fect ™ reagent was used to deliver siRNA in vivo to silence the NTS2 gene.

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
1Physiology and Biophysics, Univ. Sherbrooke, Sherbrooke, PQ, Canada; 2Integrated DNA Technologies Inc., Coralville, IA2007

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

Tuesday, May 22, 2007

i-Fect and Nav1.8 Gene Silencing in vivo

Referenced in United States Patent 20070105806

Link to patent: Free Patents Online
[0241] The effect of siRNAs against Nav1.8, formulated with iFECT, on complete Freund's adjuvant-induced tactile hypersensitivity was evaluated in rats (FIG. 5). Adult male Sprague-Dawley rats received an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs against Nav1.8 were then administered by intrathecal bolus to the lumbar region of the spinal cord on days 1, 2 and 3; specifically, for each bolus injection, 2 ug of siRNA was complexed with iFECT transfection reagent (Neuromics, Minneapolis Minn., USA) at a ratio of 1:4 (w:v) in a total volume of 10 uL. Five groups of rats (with 5 rats per group) were treated with either siRNA (AL-DP-6049, AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), or PBS, in the presence of iFECT. Tactile hypersensitivity was expressed as tactile withdrawal thresholds which were measured by probing the hindpaw with 8 calibrated von Frey filaments (Stoelting, Wood Dale Ill., USA) (0.41 g to 15 g). Each filament was applied to the plantar surface of the paw. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength and calculated with a Dixon non-parametric test (see Dixon, W. J. (1980) "Efficient analysis of experimental observations" Annu Rev Pharmacol Toxicol 20:441-462; Chaplan, S. R., F. W. Bach, et al.(1994) "Quantitative assessment of tactile allodynia in the rat paw" J Neurosci Methods 53:55-63). Tactile thresholds were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles. In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-6209, tactile thresholds were nearly normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6209, is efficacious in vivo against inflammation-induced hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-6217, resulted in the average tactile threshold trending towards baseline, with one of five rats demonstrating a normal tactile response. AL-DP-6049 and AL-DP-6218 did not significantly alter tactile thresholds compared to PBS treatment, in this experimental paradigm.[0242] These results demonstrate that siRNAs targeting Nav1.8, formulated with transfection reagent and administered intrathecally, alleviate CFA-induced tactile hyperalgesia, and therefore represent a novel approach to providing effective treatment of clinical inflammatory pain.

Monday, April 2, 2007

Magnetically Enhanced siRNA Transfection

We have added a new set of products that magnetically drive transfection.

MATra products are designed to enhance the transfection capabilties of our i-Fect and pn-Fect kits.

The technique requires lease or purchase of the Universal Magnetic Plate AND for i-Fect and other siRNA transfection reagents, please also purchase the MATra-siRNA Reagent. For pn-Fect and other Nucleic Acid transfectants, you would buy the MATra-A Regeant.
If your cells are in suspension, you will also need MATra-s-Immobilizer.
Manual for All MATra Products

Magnet Assisted Transfection Animation


Bertram, J. (2006) MATra - Magnet Assisted Transfection: Combining Nanotechnology and Magnetic Forces to Improve Intracellular Delivery of Nucleic Acids. Current Pharmaceutical Biotechnology 7, 277-285.

Goulimari, P., Kitzing, T.M., Knieling, H., Brandt, D. T., Offermanns, S.
and Grosse, R. (2005) G?12/13 Is Essential for Directed Cell Migration and Localized Rho-Dia1 Function. J. Biol. Chem., Vol. 280, Issue 51, 42242-42251.

Kumbrink, J., Gerlinger, M., and Johnson, J.P. (2005) Egr-1 Induces the Expression of Its Corepressor Nab2 by Activation of the Nab2 Promoter Thereby Establishing a Negative Feedback Loop. J. Biol. Chem., Vol. 280, Issue 52, 42785-42793.

Liman, J., Ganesan, S., Dohm, C.P., Krajewski, S., Reed, J.C., Bähr, M., Wouters, F.S. and Kermer, P. (2005) Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity. Molecular and Cellular Biology, Vol. 25, Issue 9, 3715-3725.

Luo, W., Wang, Y., Hanck, T., Stricker, R. and Reiser, G. (2006) JAB1, A Novel Protease-Activated Receptor-2 (PAR-2) - Interacting Protein Is Involved in PAR-2-Induced Activation of AP-1. J. Biol. Chem. Papers in press. Published on January 12, 2006 as Manuscript M510784200.

Tuesday, February 6, 2007

Duration of siRNA studies-pain study example

We are working with an investigator on using RNAi to relieve pain and his study prompted some discussion about the length of time before an effect is seen. It is important to note that these types of experiments take time to see an effect. In studying the pain response, the effect may take even longer.

Dorn et al (Nucleic Acids Res. 2004 Mar 16;32) examined the ability of siRNA to relieve chronic neuropathic pain targeting P2X3 in vivo. They showed a significant effect on the ability of the siRNA to relieve the pain; however there was no significant effect until around 6 days post.

Another study by Hemmings-Mieszczak et al (Nucleic Acids Research, 2003, Vol. 31, No. 8 2117-2126) showed that in an in vitro model, oligofectamine-mediated transfection of siRNA resulted in 60±90% downregulation of P2X3, but only after about 3 days.

So a key factor in any siRNA transfection study is to carry the experiment out long enough, which in in vivo work can be longer than for in vitro studies. As Mark points out, "Normally in cell culture we would look at mRNA levels at 24 h and protein levels at 48-72 h post transfection, depending upon the cell division rate and the half life of the protein. Long lived proteins in non-dividing cells could theoretically take multiple administrations of reagent over a week or more to knock down your targeted protein in vivo."

Best regards,


Monday, January 15, 2007

Transfecting Primary Cells

We welcome any comments on you experiences with transfecting primary cell lines with cationic lipids and/or electroporation.

Here're are interesting comments of transfecting primary cells from Dr. Mark Behlke, CSO of IDT:

"Just wanted to give you a project update. We have good transfection conditions worked out for LTK cells and CHO cells now. Different reagents and different conditions proved to be optimal for each cell line.

The LTK cells proved to be a real nightmare. We were testing out different siRNAs and had a weird problem that it seemed that none of the siRNA worked even a little, with the weird observation that our controls were “very low”. After quite a bit of investigation, we finally figured out that the LTK cells were triggering an IFN pathway response to the siRNAs. Unfortunately, it was a bit odd and it took some time to figure out what was going on. The cells did not just “die” or show changes in the usually time frame expected for Type-I IFN responses, but clearly were activating new cytokine related transcription units. The promoter driving your transgene is regulated by the transcription factors induced, such that NTS1 mRNA levels increased upon transfection. The controls were “low”, showing basal levels, and the transfected cells were all “high”. It turns out that our dye-labeled transfection control sequence was triggering the pathway in a sequence-specific fashion. We are now leaving out the transfection control and things are OK. We can also totally evade the response using 2’OMe modified duplexes (we usually use these modified duplexes for siRNA intended for IV administration, since exposure to PBMCs usually triggers any/all possible IFN responses).

We can avoid these responses and now have the assay system working. So, we can finally start proper testing!"

Preliminary Results from Dr. Helen Hellmich of UTMB:

"It looks like the I-fect delivered the control labeled IDT oligos to approximately the right place in the rat hippocampus 48 hours post-injection. After my research assistant comes back from her vacation, we will try a longer time point, 72 hours and alter our stereotactic coordinates slightly to more directly target the CA3 subregion. After that we will try the real siRNA injections with I-fect, hopefully in the next few weeks. I will keep you all posted to whether this really works."

Friday, January 12, 2007

Protocols for Delivering siRNA to Neurons

Here's a link to the protocol: Transfecting Schwann Cells with i-Fect and below are related data.
Figure:si RNA-mediated suppression of target gene expression in Schwann cells. A, Detergent extracts of siNeg- or siGly1- transfected Schwann cells were digested with heparitinase and subjected to immunoblot analysis with anti-glypican-1 antibodies (top);aliquots of undigested extracts wereimmunoblotted with anti-actin antibodies (bottom) to verify equal sample loading. B, Cell surface expression of glypican-1 was assessed by immunofluorescent staining of transfected cells 48 h after transfection using anti-glypican- 1 antibodies (green) and DAPI (4',6'-diamidino-2 phenylindole) to stain nuclei (red). C, Schwann cells were transfected with siNeg or si 4(V)and conditioned media and cell lysates were harvested 48 h later (left) or at the indicated times after transfection (right);aliquots of medium (top) or cell lysates (bottom) were subjected to immunoblot analysis and stained with anti- 4(V) collagen (top) or anti- -actin (b ottom) antibodies.