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

Dr. Philippe Sarret Team and Potential New Pain Targets

Shedding Light on New Pain Pathways

There is no joy in Painville. Our answer to pain is: “make it go away”! It spoils quality of life. The socio-economic costs for treatments, loss of productivity and absenteeism, are measured in billions USD$.

Today, moderate to severe pain is treated mostly with NSAIDs, narcotics or tricyclics (anti-depressants). Properly prescribed, these effectively alleviate pain. However, for cases of sustained chronic pain, they become problematic. More than 30% of the population coping with chronic pain are insensitive to morphine derivatives or other pain treatments. They can lose their effectiveness (tolerance), most can be abused and are addictive (dependence), but overall, given in multitherapy, their side effects are additive and deleterious. These problems arise from a lack of comprehension in their mode of action. This is not good news for neuropathic and chronic pain sufferers looking for long term relief.

Research that could lead to discovery of non-narcotic drugs signaling via opioidergic-independent pathways is part of the solution for people coping with chronic pain. This brings us to our back story featuring Dr. Philippe Sarret and his Research Team at the University of Sherbrooke.


About Dr. Philippe Sarret

-Masters (biochemistry), University of Nice in 1994.

-Diploma (DEA, cellular and molecular biology), University of Nice 1996.

-PhD (pharmacology), Institute of Molecular and Cellular Pharmacology, Sophia Antipolis 2000

-Post-doctorate (Neuroscience), Montreal Neurological Institute (MNI), McGill University, Montreal 2004.

-Professor, Faculty of Medicine and Health Sciences, University of Sherbrooke in 2004 -present

Sarret Website-In English

Sarret Website-In French

Tél.: (819) 820-6868, poste 12554
Téléc.: (819) 820-6887
Courriel: Philippe.Sarret@USherbrooke.ca

I asked Dr. Nicolas Beaudet, a Sarret lab member, why he joined the lab. He said, “ Philippe is a great communicator. He has the ability to articulate his complex research in a way that is easy to understand, visionary and exciting”. The aspect that Nicolas finds most intriguing is the systems approach that Philippe and the team take in understanding the mechanisms of pain. This enables them to work at them molecular level up to the whole animal. This is a key step in finding potential new pain therapies.

Drilling Down

Philippe and his team centered their efforts on G Protein Coupled Receptors (GPCRs) such as apelin, chemokines and neurotensin. As a common point, they were all recently identified in the central nervous system to provide a potential role in pain modulation.

Lately, the focus has been on the roles of Neurotensin Receptor 1 (NTS1) and Neurotensin Receptor 2 (NTS2). Recent studies have highlighted the role of these receptors in pain modulation and more is to come…:

  • Geneviève Roussy, Marc-André Dansereau , Louis Doré-Savard, Karine Belleville, Nicolas Beaudet, Elliott Richelson and Philippe Sarret. Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model.Journal of Neurochemistry 105: 1100 – 1114
  • Sarret, P, Perron, A, Stroh, T and Beaudet, A (2003). Immunohistochemical distributionmof NTS2 neurotensin receptors in the rat central nervous system. J Comp Neurol 461: 520–538.
  • Sarret, P, Esdaile, MJ, Perron, A, Martinez, J, Stroh, T and Beaudet, A (2005). Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors. J Neurosci 25: 8188–8196.
  • Dobner, PR (2006). Neurotensin and pain modulation. Peptides 27: 2405–2414.
  • Maeno, H, Yamada, K, Santo-Yamada, Y, Aoki, K, Sun, YJ, Sato, E et al. (2004). Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception. Brain Res998: 122–129.      

The wow factor for me was the clever way Philippe and his team used a new technology of 27mer NTS2 Dicer Duplex siRNA (DsiRNA) delivery in vivo as a proof for the potential of DisRNAs-based pain therapies.

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.

What Happened

Using an acute pain model, anti-nociceptive effects of NTS2, induced by a selective agonist, were significantly reduced following NTS2 silencing This resulted in rats showing an increased sensitivity to pain. By day four, the knockdown effects showed a decrease with the NTS2 function returning to normal.

What ‘s next

So we have a great start. We know that agonists binding to NTS2 in the CNS lead to analgesia. We know that DsiRNA can be used to alter the expression of this gene in vivo. We have provided a key step in learning how the NTS2 receptors can be manipulated to block pain. However, now we need to unravel the underlying mechanisms explaining these spinal analgesic properties.

It is my hope that Philippe and his team are appropriately funded. This would catalyze further discoveries in how expression of G Protein Coupled Receptors like NTS1, NTS2, APJ, CCR2 can be targeted to modulate pain. By using rodents, the team can develop tools like DsiRNA to increase the potency and duration of pain blockade. Moreover, potential toxicity and side effects need to be addressed in order to move forward towards clinical studies. These pre-clinical models prove invaluable in taking the step to studies in humans.These therapies hold the promise of providing relief for chronic pain (neuropathic, arthritic, diabetic, cancer pain, etc.) sufferers without the current side effects. Stay tuned as I will be reporting the good news as it unfolds.

Making Gains on Pain

Nicolas Beaudet

Nicolas Beaudet

Neuromics has been helping researchers make “gains on pain” from day one. Our initial sales were Opioid Receptor Antibdies licensed from Dr. Robert Elde’s lab at the University of Minnesota.

From this start, we have expanded our expertise and products. Our reputation in this area has resulted in our forming collaborations and friendships with research teams doing important work in chronic and nociceptive pain research.

Over the past several years, I have worked closely with Dr. Nicolas Beaudet, a member of Dr. Philippe Sarret’s team, at the University of Sherbrooke”. In our next backstory we will feature the vangaurd work they are doing on the Neurotensin (NTS) Receptors and pain.

Manipulation of these receptors-NTS-1, NTS-2 and NTS-3 could represent a therapy for pain independent of the opioid pathway. This means pain could be treated without narcotics meaning a reduction of side effects from current treatments including addiction to pain killers.

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.