Delivering 27mer DsiRNAs to Mice DRGs

I have been a proponent of using 27mer DsiRNAs (Dicer Substrate Small Interfering RNAs) with our i-Fect kits to deliver siRNA to the CNS for gene expression analysis. The potency of this platform was highlighted in my profile of Dr. Mark Behlke.

It was further confirmed by in Studies conducted by Dr. Philippe Serrat and his team at University of Sherbrooke.

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); Jul;16(7):1331-9. Epub 2008 Jun 3 doi:10.1038/mt.2008.98.

Using ultra low dose of DsiRNAs complexed with Neuromics’ i-Fect , they were able to successfully reduce NTS2 gene expression by up to 86% in rat lumbar Dorsal Root Ganglia after only two intrathecal injections. This was confirmed by Western Blot and qPCR analysis.

We now have further confirmation of the capabilities of this delivery platform in a just released publication by Dr. Jeffrey Mogil and team:

Michael L. LaCroix-Fralish, Gary Mo, Shad B. Smith, Susana G. Sotocinal, Jennifer Ritchie, Jean-Sebastien Austin, Kara Melmed, Ara Schorscher-Petcu, Audrey C. Laferriere, Tae Hoon Lee, Dmitry Romanovsky, Guochun Liao, Mark A. Behlke, David J. Clark, Gary Peltz, Philippe Séguéla, Maxim Dobretsov and Jeffrey S. Mogil. The β3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test. doi:10.1016/j.pain.2009.04.028.

IT Delivery of siRNA in vivo supplement

Knockdown of rSNSR1 in vivo

The parade of success with use our i-FectTM in vivo grows. Here's the most recent study:

Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell, Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
...For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul...

Related Data:



Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.

Did Your RNAi Experiment Work?

This is a good methods publication on RNAi transfection: Reliably Validating RNA Interference with qRT-PCR.

Bill Wang, Song Tian, Qiong Zhou, and Xiao Zeng. SA Biosciences.

Intrathecal Delivery of siRNA

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

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...

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).

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.

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.