The Quest for Better Pain Therapies

G- protein coupled receptor (GPCR) and Drug Responsiveness

About Dr. Laura Bohn 

bohn1

Dr. Laura Bohn

 Background:

Spring 2009-Associate Professor (tenured) at The Scripps Research Institute, Department of Molecular Therapeutics, Jupiter, FL.

10/2007- Associate Professor (tenured), The Ohio State University College of Medicine, Departments of Pharmacology and Psychiatry, Program in Pharmacogenomics

8/2003-9/2007 Assistant Professor, The Ohio State University College of Medicine,

1/1999–8/2003 Post-Doc/Assistant Research Professor. Duke University Medical Center, Department of Cell Biology. Durham, NC. 

 

 

My company’s foundation is built on serving pain researchers. As a result, I have the good fortune of working with customers and collaborators who openly share the subtleties of their research and the future impact it could have on improving pain therapies.

Pain is complex. Today, pain therapies often fall short and are rife with unwelcome side effects. This undesrcores why I am pleased to feature Dr. Laura Bohn. She and her team are probing ways to improve  response effectiveness and reduce side effects.

 Beginnings

The story starts with Laura’s Post Doc work in Dr. Marc Caron’s lab at Duke University.  Marc in Collaboration with Dr. Dr. Robert Lefkowitz genetically engineered mice that lacked a protein switched called  “beta-arrestin 2.”  This switch is part of the opioid pathway that regulates how we perceive pain. The GPCR, muOpioid (mOR) is the primary target for narcotic pain killers, like morphine.

In her initial work, Laura found that morphine treated mice lacking the beta-arrestin2 switch swere able to tolerate  mild pain stimuli up to 3X longer than normal mice.  These mice had a higher level of sensitivity to morphine both in magnitude and duration. 

Bingo. This path for Laura’s excellent journey is now lit…understanding how the molecular regulation of G protein coupled receptors (GPCR) can translate to overall drug responsiveness in vivo.  Getting better response from lower dose is all good.

Current Work

As a researcher at Ohio State University, Laura and her team have continued to broaden and deepen their understanding of  GPCR signaling and beta-arrestin desensitivation (figure 1).

gpcr_regulation 

She is currently doing research with mice that have genetic deletions of GRKs (GRK3, GRK4, GRK5, and GRK6; heterozygotes for GRK2) and barrestin-2.

This expands the playing field. This expansion includes  studying other GCPR related pathways. Serotonin 2A receptors (5-HT2ARs), for example, are molecular targets for drug-induced hallucinations:

Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn. Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo. Published online on January 14, 2008, 10.1073/pnas.0708862105.

The conclusion: 5-HT2AR–β-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.

Future Considerations

I look for Laura and her team to continue the quest of doing more for less when it comes to novel pain and other therapies. Further success would provide the foundation for the development of therapies that would require less dosing, better response and reduced side effects.

Laura mentioned to me that further directions could involve the use of gene silencing tools like siRNA. The effects of silencing GPCR-beta-Arrestin receptors in-vivo would be an important study as it would enable she and her team to study  impact of  desensitivation on the repsonse to morphine and other drugs by normal mice.

Featuring Dr. Laura Bohn

Dr. Laura Bohn

Dr. Laura Bohn

January’s Story: Decoupling GPCR Pain Therapies from Destructive Side Effects. 

We are pleased to have Dr. Laura Bohn as our “coming soon” featured researcher. 

She caught my attention when she referenced one of our  Opioid Receptor Antibodies in the publication: C. E. Groer, K. Tidgewell, R. A. Moyer, W. W. Harding, R. B. Rothman, T. E. Prisinzano, and L. M. Bohn. An Opioid Agonist that Does Not Induce µ-Opioid Receptor—Arrestin Interactions or Receptor Internalization. DOI: 10.1124/mol.106.028258.

She, her team and callaborators are focused on an interesting and important aspect of pain therapies discovery…finding ways to de-couple the benefits of opiate based pain medications from their current side-effects including constipation, respiratory suppression and addiction.

I am looking forward to drilling into the specifics of this important research.

Making Connections that Count

Building Neuroscience Value Networks

For this month’s backstory, I am excited to introduce Dr. Ijad Madisch and the Networking Site he founded, ResearchGATE corporation (www.researchgate.net). 

Madisch, 27, an M.D. and Ph.D. virologist, says he and some friends came up with the idea when he moved from his native Germany for a research traineeship at Harvard Medical School in Boston. He found that collaborating with colleagues in Germany was awkward because e-mail wasn’t an efficient way to share updated protocols and drafts of papers. Meanwhile, sites such as Facebook and LinkedIn were taking off. “I thought, ‘This adapted to the requirements of researchers would be of big benefit to every researcher in the world,’ “ says Madisch, who is now working full-time as ResearchGATE Corp.’s CEO. The idea has become a valuable reality.

And we are happy. Having published blogs and uploaded methods/protocols into various wikis, It is easy to see the potential and power.  Users post their profiles and link up with contacts.  More importantly, they can also include information such as publications, methods, protocols and research skills. 

This provides the foundation for Neuroscience Researchers to resolve shared issues including alerting the community to new methods and approaches.  It can also work to enable “those who have done it to help those who are trying”.  This activity is the base for driving efficiencies and reducing experimental bottlenecks. In this sense, by helping others we are helping ourselves.

ResearchGATE, though launched only 6 months ago, already has 15,000+ participants. These cover many different Science Disciplines. The challenge will be to build a large network of Neuroscientists and catalyze active participation. I will be inviting select Neuromics’ Customers to participate so stay tuned.


The Sensory and Autonomic Side of Spinal Cord Injury

About Dr. Matt Ramer

Matt Ramer

Matt Ramer

  • 2001-Present-Associate Professor-University of British Columbia and ICORD
  • Post Doc-King’s College London
  • PhD.-Physiology-Queen’s College Kingston, Ontario

Matt Ramer Website

Awards and Funding

Email: ramer@icord.org

Lab Members: A. Gaudet, J. Inskip, A. Scott, L. Soril

Finding Fixes for Injured Nerves

I first became aware of Matt’s research in early 2005. This was catalyzed when he kindly shared excellent IHC images his lab generated using our BDNF and NT-3 antibodies. I was impressed with him and his team’s data and related publications. I did not understand the context of his work and the potential future impact on people suffering peripheral nerve and spinal cord injury (SCI) and wanted to learn more.

He has generously taken the time to open up my view on spinal cord injury (SCI) and what are the challenges in finding therapies and cures. I had equated success soley with restoring mobility. I knew little of the bigger complexites and problems faced by sufferers of SCI.

More than 300,000 people in the United States and Canada suffer from SCI. The ecomonic cost is in the 10s of billions. One of the horrors of SCI is lost mobility.

People with SCI also suffer from a host of problems related to loss of senory and autonomic functions. Sensory and autonomic nerves in the periperal nervous systems (PNS) connect to the spinal cord dorsally. This is different and separate from those controlling movement and motor function. A little know fact is autonomic dysfunctions represent the primary causes of morbidity and mortality following SCI.

So what are the functions that are most important to SCI patients and how should they be prioritized for basic research and drug discovery? Here are two publications that provide insight:

Kim D. Anderson, Ph.D. Targeting Recovery: Priorities of the Spinal Cord-Injured Population. October 1, 2004, 21(10): 1371-1383. doi:10.1089/neu.2004.21.1371.

J A Inskip, L M Ramer, M S Ramer and A V Krassioukov. Autonomic assessment of animals with spinal cord injury: tools, techniques and translation. Spinal Cord advances online publication 10 June 2008; doi: 10.1038/sc.2008.61

The Funding Gap

If we look through the eyes of those suffering from SCI, we know that there are a mryiad of health issues that are outside the problem of lost mobility. I fear the public including those responsible for funding define cure as “the paralyzed can walk”. This is evidenced by a gap between motor vs. sensory/autonomic research and priorities. This gap needs to be closed. The work of Matt and his colleagues represents progress and needs to be supported with funding growth. This backstory highlights how research could feed the discovery of therapies that would answer the recovery priorities of SCI pateints. They, after all, know best.

The Backstory

The story starts in 2000 and highlights Matt’s research at King’s College London. This research was done in collaboration with Dr. Stephen McMahon and Dr. John Priestly.

They showed that regeneration in damaged rat sensory neurons was possible. Injured dorsal roots, treated with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure:

Matt S. Ramer, John V. Priestley & Stephen B. McMahon. Functional regeneration of sensory axons into the adult spinal cord. Nature 403, 312-316 (20 January 2000) | doi:10.1038/35002084.

This is a tight rope act. While there is opportunity for regeneration, there are also inhibitors to nerve growth at work. Regeneration becomes more problematic as a function of time. Of the neurotrophins that promote regeneration, NT-3 appears to best at combating the competing inhibitory effects of proteins like NOGO-A. These inhibitory proteins are suspected to be secreted by astrocytes and microglia:

Matt S. Ramer, Ishwari Duraisingam, John V. Priestley, and Stephen B. McMahon. Two-Tiered Inhibition of Axon Regeneration at the Dorsal Root Entry Zone. The Journal of Neuroscience, April 15, 2001, 21(8):2651-2660.

Images: Axon growth 2 weeks after rhizotomy plus immediate NT-3 treatment. A, In intact animals, CTB-labeled terminals are present in lamina I and III, but absent from lamina II. B, Regenerating axons grow along the pial surface of the cord and in the superficial laminae of the gray matter, avoiding the degenerating cuneate fasciculus. C, Dark-field micrograph of B. Scale bar: B, 100 µm. D, Dark-field parasaggital section from a 2 week rhizotomized and NT-3-treated rat. E, Same section as in D, immunostained for CTB. CTB-labeled axons can be seen on the pial surface (arrowheads) and within the cord. Many axons have turned to grow in a rostrocaudal direction but appear to do so in the superficial laminae of the gray matter rather than the white matter. Some individual axons can be traced for up to 2 mm. F, In zones in which the density of regenerated axons is greatest, they form a longitudinal bundle in the gray matter, with few axons in the more superficial white matter (arrows). G, Many axons possess terminal swellings that may be growth cones or termination bulbs. Scale bar: E, 300 µm. The Journal of Neuroscience, April 15, 2001, 21(8):2651-2660.

Through the Looking Glass

Matt and his colleagues continue to gain understanding and refine methods for nerve regeneration. They are also studying plasticity and how these neurons connect to sensory and autonomic neurons in the PNS. This is analogous to re-wiring what was once severed. This would enable restoring of functions important to sufferers of SCI. The related good news is that even partial reconnection enable restoration of these lost functions.

Stepping through the looking glass involves understanding the specific role of these neurons. His recent works include:

Matt Ramer. Anatomical and functional characterization of neuropil in the gracile fasciculus. The Journal of Comparative Neurology. 10.1002/cne.21785.

  • Neurokinin-1 (NK 1) Receptor-Detects a band at 80-90 kDa on Western blots of membranes prepared from cells transfected with the rat substance P receptor (Vigna et al., 1994); stainingin rat spinal cord was blocked by preabsorbing the antiserum with the immunizing peptide (Mantyh et al., 1995)-Dilution 1:2,000
  • Substance P-The distribution of immunoreactivity in rat spinal cord is identical to that described previously (Hunt et al.,1981); in dual-labeling experiments, it labels the same structures as a polyclonal rabbit anti-SP (1:1,000; Peninsula/Bachem; T-4107; data not shown).

Here Matt and his team report on the morphology, inputs, projections, and functional properties of these neurons. Small fusiform and larger lentiform neurons are most abundant in the gracile fasciculus of the cervical and lumbar enlargements and are absent from the cuneate fasciculus and corticospinal tract. Many have dendrites that run along the dorsal pia, and, although in transverse sections these neurons appear isolated from the gray matter, they are also connected to area X by varicose and sometimes loosely fasciculated dendrites. These neurons receive neurochemically diverse, compartmentalized synaptic inputs (primary afferent, intrinsic and descending), half express the substance P receptor, and some project supraspinally. Unlike substantia gelatinosa neurons, they do not express protein kinase C gamma. Functionally, they have small receptive fields, which are somatotopically appropriate with respect to their anterior-posterior position along the neuraxis. They respond to innocuous and/or noxious mechanical stimulation of the distal extremities, and some are prone to central sensitization or windup. Morphologically, neurochemically, and functionally, therefore, these cells most closely resemble neurons in laminae III-VI in the dorsal horn.

Closing Thoughts

There is hope for SCI patients. It is clear that related research and funding needs to expand dramatically beyond the current narrow focus on restored motor function and mobility. The priorities are documented and understood. The story continues. Real progress will be marked by answering these priorities with restored function. Sensing pain, pressure, temperature, etc. where today there is only nothingness. Controlling autonomic functions that pose such a risk to SCI sufferers. I will continue to report the progress of Dr. Matt Ramer and his colleagues. Godspeed to them.

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.

Announcing our first story: Dr. Mark Behlke

Story to be published July 11, 2008.

Dr. Behlke, his team and collaborators are working on improving the delivery of Dicer-substrate siRNAs (DsiRNAs) in vitro and in vivo.

His work has helped establish siRNA technology as a viable tool for drug development.

Please see the article titled RNAi Researchers Galvanized by Advances: Technology’s Viability in Drug Development Is Finally Established by Elizabeth Lipp.

It appeared in the June 1, 2008 issue of Genetic Engineering & biotechnology News.

Here’s a notable quote from Dr. Behlke:

Long dsRNAs have been employed for many years as a means to modulate gene expression in plants, yeast, and C. elegans. 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.

My first “News Behind the News” story posted to this Neuromics blog will feature Dr. Mark Behlke, CSO, Integrated DNA Technologies.