In Vivo application of RNAi to study pain

This overview is from 2010. I am posting a link because it undercores the need to have transfection reagents that have the ability to deliver small doses of siRNA in vivo.
"One of the biggest challenges in using RNAi in pain research is delivery of siRNA to the CNS in sufficient concentrations. This obstacle exists because siRNA by itself does not cross the blood brain barrier (BBB) and is degraded in the blood by endonucleases. Intravenous or oral administration is, therefore, inadequate to achieve desired protein knockdown. The use of transfection agents and intrathecal delivery has enhanced siRNA uptake by target tissues in recent studies." Zachary J Clark, Gurwattan S. Miranpuri, Daniel K Resnick. In Vivo application of RNAi to study pain. Annals of Neurosciences, Volume 17, Number 3, July 2010.

Please click through the link and you will learn of techniques currently used to delivery siRNA in vivo for pain research.

siRNA Delivery Group on Linkedin

I wanted to make readers aware of an excellent discussion group on Linkedin named "siRNA Delivery". Included are tip, updates on commercialization and key publications.

Here're some examples:

• Alnylam Announces Publication of Pre-clinical Results with ALN-HTT, an RNAi Therapeutic for the Treatment of Huntington’s Disease, in Experimental Neurology
• Delivering siRNA to Neurons
• Life Technologies develops new drug delivery technology
• Recent experiments demonstrate a novel method of liposomal encapsulation of siRNA/RNA/DNA at 4C, could be a promising method to minimize degradation.

Happy reading.

Using MATRa for siRNA Transfection of Carcinoma Cell Lines

MATRa^TM -Magnet Assisted Transfection is an easy-to-handle, very fast and highly efficient technology to transfect cells in culture with siRNA. Multiple successes with the system includes Carcinoma Cell Lines.

Efficient transient transfection of siRNA in head and neck cancer cells. The cell line ANT-1 was transiently transfected with MATra-A (1 µl/1 µg DNA) in a 6 well format (5 x 105 cells/cavity) with siRNA against protein 1 (100 nM). After 24 hours total RNA was isolated and expression of protein 1-specific mRNA determined by RT-PCR (upper lane). SiRNA 13 are three different oligonucleotide sequences. Control for consistent loading and cDNA quality: expression of ubiquitary GAPDH mRNA (lower lane).
Protein 2 expression in head and neck cancer cells GHD-1. GHD-1 cells (5 x 105 cells/cavity of a 6 well plate) were transiently transfected with two different siRNAs against protein 2. Expression of protein 2 was detected with specific antibodies in an immunoblot 72 hours after transfection with MATra-A (1 µl / 1 µg DNA). As control ubiquitary β-actin was detected as well.
Treating the carcinoma cells with specific siRNA caused a clear inhibition of protein 1/protein 2 expression which indicates high transfection efficiencies.
(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany).

"After having tested MATra in a variety of experimental set ups we can summarize the following advantages: High transfection efficiency   Easier to handle   High reproducibility Serum compatibility Low sensibility against cell confluence" Dr. Oliver Gires, LMU Munich, Germany

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...

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.

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.