Tuesday, December 16, 2008

Intrathecal Delivery of siRNA

We wanted to present yet another publication referencing successful delivery of siRNA using i-FectTM:

Suneeta Tumati, Tally Largent Milnes, Henry I. Yamamura, Todd W. Vanderah, William R. Roeske and Eva V. Varga. Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. doi:10.1016/j.ejphar.2008.10.033

...siRNAs stock solutions (100 μM) were prepared in double distilled RNAse free water and stored in aliquots at −80 °C. For intrathecal treatment, aliquots of the stock solution (2 μg of the appropriate siRNA) were mixed (1:5 v/v)with i-Fect transfection reagent (Neuromics, Edina, MN). After recovery from the surgery (5–7 days), the animals received intrathecal injections (2 ug siRNA/10 ul/rat) of either a lipid encapsulated Raf-1-selective siRNA mixture (Smart pool siRNA, Dharmacon Inc; Chicago, IL, Cat # L-087699-00) (Raf-1 siRNA groups) or i-Fect encapsulated non-targeting dsRNA (Dharmacon, #D-001810-01-20) (control mismatch siRNA groups) or the transfection lipid alone, once daily, for 3 days, as described earlier (Gardell et al., 2002). Intrathecal injections of the siRNAs or the transfection agent alone did not cause any sign of behavioral toxicity. Western blots, using a Raf-1-selective antibody, indicated that intrathecal treatment with the Raf-1-selective siRNA mixture for 3 days significantly reduced Raf-1 protein levels in the dorsal root ganglion and in the dorsal horn of the spinal cord...

Sunday, November 2, 2008

Delivering Naked siRNA by Direct Injection

This blog has featured methods for delivering siRNA in vivo by intrathecal injections. These methds all highlight conjugation with novel cationic lipid based carriers like i-Fect ™. Here we highlight delivery by direct injection:

Dr. Matthew Farrer and his team, including researchers from Alnylam, have successfully demontrated the delivery of naked siRNA by direct injection. In this study, they delivered chemically modified murine and human alpha-synuclein (SNCA) siRNAs to the hippocampus by direct injection resulting in silencing of gene expression.

To learn more access:

In vivo silencing of alpha-synuclein using naked siRNA Jada Lewis, Heather Melrose, David Bumcrot, Andrew Hope, Cynthia Zehr, Sarah Lincoln, Adam Braithwaite, Zhen He, Sina Ogholikhan, Kelly Hinkle, Caroline Kent, Ivanka Toudjarska, Klaus Charisse, Ravi Braich, Rajendra K. Pandey, Michael Heckman, Demetrius M Maraganore, Julia Crook, Matthew J Farrer. Molecular Neurodegeneration 2008, 3:19 (1 November 2008).

Sunday, October 19, 2008

ACIC3 Receptors Knockdown in vivo

Researchers using siRNA complexed with our i-Fect ™ transfection regent have successfully knocked down ASIC3 Receptors in vivo. This publication joins the growing parade (starting with Luo et al, 2005) that refererence successuful modulation of receptors involved in pain using siRNA complexes.

These studies all share animal behavior studies showing a marked change in response to pain stimuli after treatment.

In this study, Dr. Eric Lingueglia and his team found Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Emmanuel Deval, Jacques Noël, Nadège Lay, Abdelkrim Alloui, Sylvie Diochot, Valérie Friend, Martine Jodar, Michel Lazdunski and Eric Lingueglia. ASIC3, a sensor of acidic and primary inflammatory pain. The EMBO Journal advance online publication 16 October 2008; doi: 10.1038/emboj.2008.213

Cy3-labelled siRNA no. 1121 and its corresponding scramble (no. 1121S; GCTCACACTACGCAGAGAT) synthesized by MWG Biotech (Germany) were injected in rats by intrathecal bolus to the lumbar region of the spinal cord once a day for 3 days before the induction of inflammation with CFA. Each 10-ml injection corresponded to 2 mg of siRNA complexed with i-Fect siRNA transfection reagent (Neuromics) at a ratio of 1:4 (w:v) (Luo et al, 2005), following the supplier’s suggested protocol. siRNA uptake in lumbar DRGswas monitored by fluorescence microscopy on cryostat sections 24 h after a single intrathecal injection.

Here’s a synopsis of results:
Inflammation was produced by CFA injection, which led to primary heat hyperalgesia, and this hyperalgesia was drastically reduced by the ASIC3 blocker APETx2 injected subcutaneously, which only access cutaneous nociceptors. It was also drastically reduced when, before triggering the inflammation state, intrathecalinjections of an siRNA against ASIC3 had induced a knockdown of ASIC3 expression in lumbar DRGs.

Saturday, October 4, 2008

siRNA-mediated gene silencing

Dr. Josephine Lai (Professor of Pharmacology, University of Arizona) is a pioneer in developing experimental designs and methods for delivering siRNA to the CNS for gene expression analysis.She and her team have documented these in the publication:

Modulating Sensory Systems Using RNAi(pdf - 187Kb)© 2007 Lai

For researchers desiring to effectively deliver siRNA to the CNS for gene expression analysis of specific receptors, this publication offers proven methods. These include:

  • The Choice of siRNA

  • Choosing and Optimizing Transfection Reagents for siRNA Delivery to the Nervous System
  • Delivery Systems-Microinjection and Infusion (using mini-osmotic pumps)
  • Validation

We will continue to track advances by Dr. Lai and team.

Thursday, August 28, 2008

Transfecting Sympathetic Neurons

GDNF and Ret are important to the growth, maintenance and survival of Neurons. The GDNF ligands act via activation of Ret. Every step towards understanding the intricacies of this pathway, brings researchers closer towards unlocking the code for Neurodegenerative Disease therapies.

siRNA is an important tool for studying the Neurotrophic pathways as researchers can use it to modulate the expression of related receptors. The tricky part is getting sufficient siRNA into neurons to do the appropriate studies of how modulating targeted genes results in changes in protein expression.

Here Drs. Cynthia Tsui and Brian Pierchala have published results from there studies of C2AP and Cbl-3/Cbl-c and Ret Transduction. One of the keys to this study was using siRNA to silence CD2AP and Cbl-3 expression. By turning these off they were able to identify a critical checkpoint in the Ret pathway.

Cynthia C. Tsui and Brian A. Pierchala CD2AP and Cbl-3/Cbl-c Constitute a Critical Checkpoint in the Regulation of Ret Signal TransductionJ. Neurosci., Aug 2008; 28: 8789 - 8800 ; doi:10.1523/JNEUROSCI.2738-08.2008.

...Control, CD2AP, and Cbl-3 siRNAs (Applied Biosystems/Ambion) were transfected into 4 DIV sympathetic neurons using the i-Fect ™ reagent according to the manufacturer’s instructions (Neuromics). Transfection efficiency was determined by the cotransfection of a fluorescently labeled nontargeting, control siRNA (siGLO RISC-free siRNA; Dharmacon RNA Technologies). Immunoblotting of the targeted proteins determined that the maximal knockdown of protein expression was observed 72 h after siRNA transfection. Greater than 90% of SCG neurons were transfected, as ascertained by the level of intracellular fluorescence of the siGLO siRNA.

Sunday, August 24, 2008

Down Regulating the Smad and Neuro-regeneration

The inventors down regulated Smad 2/3 (an inhibitor of neuro-regeneration) in vivo via delivery of siRNA to the spinal cord using catheters.

Inhibiting smad signaling promotes neuron regeneration.
Inventors: Fan Wang, Zhigang He
USPTO Application #: 20080031911
Inhibition of Smad2/3 Signaling Promotes Axonal Regeneration after Spinal Injury in Rats-Gene Expression Knockdown in vivo!
One hour after the spinal cord is lesioned, the rats in the SB-505124 group receive a bolus injection of SB-505124 (30 mg/kg) in 0.9% saline administered via a tail vein. The treatment is repeated every 24 hours on days 1 through 7 post-lesion. Vehicle only control rats undergo the same treatment but are injected with an equal volume of 0.09% saline in a tail vein. At the same treatment time points as the SB-505124 group, the Smad2/3 siRNA group and corresponding controls receive 10 .mu.l rat Smad2/3 siRNA (Dharmacon, Lafayette, Colo.), mismatch siRNA, or transfection reagent only delivered to the spinal cord via the catheters. The siRNA (or mismatch siRNA control) complexes are prepared immediately prior to administration by mixing the RNA solution (200 .mu.M in annealing buffer) with a transfection reagent, i-Fect ™ . (Neuromics, Edina, Minn.), in a ratio of 1:4 (w:v). At this ratio, the final concentration of RNA as an RNA/lipid complex is 2 .mu.g in 10 .mu.l.

Thursday, July 31, 2008

siRNA and Diabetes

I'll be keeping my eyes on this and post updates.

Quark Pharmaceuticals Announces First Patient Dosing by Pfizer in Phase II Trial of RNAi Therapy in Diabetic M
Clinical Program Leverages Quark's RNAi Technology FREMONT, Calif., July 30 /PRNewswire/ -- Quark Pharmaceuticals, Inc., a development-stage pharmaceutical company discovering and developing novel RNA interference (RNAi)-based therapeutics,...

Sunday, July 20, 2008

Improving 27mer DsiRNA Performance

The DsiRNA story marches forward with yet another important publication. We will give you an opportunity to upload the fulltext article at the end of this posting.

Dr. Mark Behlke, Dr John Rossi and team have been gaining deeper understanding of the Mechanism of Dicer-substrate small-interfering RNA (DsiRNA) processing. This understanding is leading to better and better designs of the RNA duplexes. These designs or chemical modifications are necessary steps in the drug design and development process.

This publication looks at design from the perspective of:

Nuclease Stability
immune response

I believe this is an important publication for researchers wanting to better understand:

The mechanisms behind successful delivery of DsiRNA for gene expression studies.
Variations in potency.

upload article: oligo-18-p187-2008-collingwood-dsirna-modifications1

Tuesday, July 8, 2008

Coming Soon

Agenda Topics:
Target Discovery and Validation
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
  • Wednesday, June 18, 2008

    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.”

    Monday, June 16, 2008

    Wednesday, June 4, 2008

    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.

    Wednesday, April 9, 2008

    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.