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  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

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 reference successful 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 DRGs
was 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, intrathecal
injections of an siRNA against ASIC3 had induced a knockdown of ASIC3 expression in lumbar DRGs.

I will continue to publish updates.

The First Story is Here!

Dr. Mark Behlke and 27mer DsiRNAs

 

I am pleased to be featuring Dr. Mark Behlke’s story as our first. This was an easy choice because our main characters, Mark and the 27mer DsiRNAs (Dicer Substrate Small Interfering RNAs), are rising stars in small interfering (siRNA) based research.

 

siRNAtechnology addresses the need for Biosciences Researchers and Clinicians to selectively reduce expression in genes of interest. If effectively delivered, these siRNAs act as “dimmer” or “off” switches for gene expression (gene silencing). Traditionally, synthetic 21mer RNA duplexes have been employed to trigger RNA interference, a method that was pioneered by Tuschl and colleagues in 2001.

 

I became interested in Mark’s work in 2003. Our collaboration was catalyzed by Neuromics’ need to provide our customers better ways to deliver siRNAs to neurons in vitro and in vivo using our i-Fect ™  transfection kits. Successful outcomes for our customers hinged on the potency and duration of gene silencing. In short, our customers needed potent knockdown reagents and optimized ways to deliver these reagents to neurons, both in vivo and in vitro.

 

Mark has gone above and beyond the call of duty in addressing this need. His investment of time and his company’s resources (Integrated DNA Technologies) has proven to be a linchpin in successful Neuroscience Research outcomes and has resulted in exciting publications for several of our key customers.

About Dr. Mark Behlke

 

Dr. Mark Behlke is the Chief Scientific Officer (CSO) at Integrated DNA Technologies (IDT) and has been directing R&D activities of their Molecular Genetics & Biophysics research groups since 1996.  Dr. Behlke (with Dr. John Rossi, from the Beckman Research Institute at the City of Hope) is a scientific co-founder of Dicerna Pharmaceuticals.  Previously, Dr. Behlke was a HHMI Physician Postdoctoral Fellow at the WIBR in the laboratory of Dr. David Page and a Resident Physician in Internal Medicine at Brigham and Women’s Hospital, Boston.  He received his MD/PhD degrees from Washington University, St. Louis in 1988, where he studied immunogenetics in the laboratory of Dr. Dennis Loh.  He received his B.S. degree from the Massachusetts Institute of Technology in 1981.

 

Contact information:

Mark Behlke M.D., Ph.D,Chief Scientific Officer

 

Integrated DNA Technologies, Inc.

1710 Commercial Park

Coralville, IA  52241

USA

 

800-328-2661

319-626-8432 office

319-626-9621 fax

mbehlke@idtdna.com website: http://www.idtdna.com/


My goal here is to spread the story of 27mer DsiRNAs. This technology has proven an effective tool for my Neuroscience Research Customers. With continued development, this could become a cornerstone of functional genomics.
                          

The Back-story 

Where it starts

A lot has to happen right for siRNA to reduce expression of mammalian genes. The siRNA molecules must first   be transfected into the cells of interest. Once inside, they must be correctly processed by the cells’ biochemistry

Our story starts with Mark’s curiosity concerning siRNA length and what happens to these molecules inside the cell. The idea was to systematically study the effects of varying siRNA length on triggering gene silencing. This project was done in collaboration with Dr. John J. Rossi (Beckman Research Institute) and other members of his lab at the City of Hope National Medical Center (most notably Dr. Dongho Kim, a postdoc in the Rossi lab).

The team knew that mammalian cells use a Dicer complex to process longer length dsRNAs into functional 21mer siRNAs and then feed these into a complex called “RISC” (RNA induced silencing complex).   

Long RNAs (several hundred bases) can be introduced into worms or flies and trigger RISC. 

In mammals, the introduction of similar long RNAs triggers immune responses and cell death Use of small 21mer siRNAs mostly avoids this problem and permits use of RNAi in mammals This traditional approach made sense given the siRNA-Dicer-RISC pathway (fig. 1). The team looked at the effects of transfecting into cells synthetic dsRNAs ranging in length fom 21mers to 30mers

 

Fig. 1: Pathways in siRNA .  Long vs. short dsRNAs are differentially processed as shown.

What happened? Was 21mer length optimal?

Their findings were quite unexpected: they observed that synthetic RNA duplexes 25–30 nucleotides in length could be up to 100-fold more potent than corresponding 21mer siRNAs. Why?  The 27mers were later shown to be a substrate for Dicer, and were processed down to 21mer size. Drs. Rossi and Behlke theorize that increased potency may result from forcing the system to interact with Dicer, which then invokes a natural RISC loading pathway that is denied to 21mer RNAs.  The 27mers “primed the Dicer pump”, resulting in better access of the 21mer product for RISC.

This meant that less siRNA would be needed for gene silencing – i.e., that the RNAs were more potent and could be used at lower dose. Important for many reasons among them less toxicity and lower research expense.

Please see: Dong Ho Kim, Mark Behlke, Scott Rose, Mi-Sook Chang, Sangdun Choi & John Rossi. Synthetic dsRNA Substrates Enhance SiRNA Potency and Efficacy  Nature  Biotechnology. Published online 26 December 2004;doi10.1038/nbt1051.

The rest of the story

Great news! The 27mers were more potent and could prove a better tool for Researchers studying gene function. It’s never that easy. While potency of the 27mer DsiRNAs proved greater than the 21mers in many assays, Mark shared that results proved frustratingly unpredictable depending on the target. More insight was needed.

As Mark and the team gained more experience by targeting additional sites in other genes, examples were found where the 27mer DsiRNAs had greater, the same or less potency than 21mers siRNAs for the same site. This wide variation in performance resulted from differences in dicing patterns: sometimes Dicer processing resulted in a “good” 21mer product for RISC and sometimes resulted in “bad” products.

The root cause of this unpredictability proved to lie in the design of the synthetic 27mers. The original designs were blunt ended (both ends) and Dicer processing was unpredictable – essentially random – and the precise 21mer cleaved out of the 27me parent varied from sequence to sequence. This forced the team to learn how to design better 27mers that have predictable Dicer cleaving patterns.  The new improved design is a 27mer asymmetric duplex having a single 2-base 3’-overhang on one end and 2 DNA bases on the opposing blunt end.

 

Rose SD, Kim DH, Amarzguioui M, Heidel JD, Collingwood MA, Davis ME, Rossi JJ, Behlke MA. Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res. 2005 Jul 26;33(13):4140-56. Print 2005. PMID: 16049023

 

Also  see: 27mer RNA Duplexes as Triggers of RNAi. Exploiting the Biochemistry of Dicer. BIOforum Europe 06/2006, pp 25–27, GIT VERLAG GmbH & Co. KG, Darmstadt, Germany.

 

 

The proof

 

So now we have optimal 27mer DsiRNAs, let’s put them work in the CNS with i-Fect ™ .

 

IDT and Neuromics collaborated with Philippe Sarret at the University of Sherbrooke Neuroscience Center. Philip and his teamed selected Integrated DNA Technologies’ designed 27mers DsiRNAs and i-Fect as core research tools for their proof of concept. They wanted to prove that an RNAi approach could be used to study pain pathways in rats in his lab by selective knockdown of specific CNS receptors via direct injection of DsiRNA (formulated in i-Fect) into the spinal cord of rats.

 

Their recently published findings were remarkable.

 

Please see: 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.

 

Low dose DsiRNA (0.005 mg/kg) was highly effective in reducing the expression of the Neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, the results showed a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3–4 days after administration of DsiRNA.

 

In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), the team also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor.

 

Mark’s Vision

 

This story has no end point because the key players are continuing to collaborate and march forward on their journey of discovery. Mark said it best, “Discovering new stuff is why I do what I do. It’s nice if the findings are interesting, but it is better if it has the potential to impact the world and improves people’s lives in some way.”  The basic biology studied now may lead to new generations of drugs tomorrow that treat problems that cannot be effectively treated today.

 

The good news is most of the story lies ahead. In fact, Biotech Companies are being formed and funded on the promise of 27mer DsiRNAs’ potential both as a platform for drug development and as actual therapeutics.  For an example, please visit Dicerna Pharmaceuticals.

 

Who knows… someday, 27mers DsiRNAs could be the key for curing Neurodegenerative and other Diseases. Stay tuned.