Scripps Florida Scientists Awarded $3 Million to Develop New, More Effective Pain Treatments

We profiled Dr. Laura Bohn research in one of our news stories. We are excited to share the news.Dr. Laura Bohn

JUPITER, FL, February 29, 2012 – Scripps Florida scientists have been awarded $3.1 million by the National Institute on Drug Abuse, part of the National Institutes of Health, to study and develop several new compounds that could prove to be effective in controlling pain without the unwanted side effects common with opiate drugs, such as morphine, Oxycontin®, and Vicoden®.

Laura Bohn, an associate professor in the Department of Molecular Therapeutics and Neuroscience at Scripps Research, and Thomas Bannister, an assistant professor in the Department of Chemistry and associate scientific director in the Translational Research Institute at Scripps Research, will serve as joint principal investigators for the new five-year study.

Their study will focus on four new classes of compounds that appear to differ fundamentally from opiates inthe side effects that they can produce.

“Once we more fully understand how these compounds work, we expect to optimize and develop them as novel drugs,”said Bohn. “We hope to produce potent pain relievers without the problems associated with current treatments.” Full article: http://www.scripps.edu/news/press/20120229bohn-bannister.html

We wish her great success in her research aimed at discovering improved solution for managing pain.

Featuring Dr. Pat Carr

Amyotrophic Lateral Sclerosis (ALS)-New Twists on Root Causes

Teacher, Mentor and Friend    Dr. Pat Carr has been a key figure in helping shape the direction of my company. He has a gift for communicating the nuances of his research and coaching me on how to best serve labs like his. Based on these interactions, it came as no surprise to learn of his being Recognized for Excellence in Teaching, Research and Service at University of North Dakota.

“Dr. Carr has a magic way of teaching,” said second-year medical student, Tyson Bolinske. “He is able to take the most difficult topics and, through detailed notes, logically break down the material.

From a recent dialog, I learned of his growing work on the Ventral Horn and search for root causes of Amyotrophic Lateral Sclerosis (ALS).   I wanted to learn more! I would like to thank Pat for agreeing to share his story and giving me the opportunity to feature highlights in  “News Behind the Neuroscience News”.

 Information on ALS

ALS is an insidious disease.  It is a progressive neurodenerative disease that is always fatal. Approximately 5600 new cases are diagnosed each year. Average survival is typically 3-5 years from onset. The most common form of ALS in the United States is “sporadic” ALS. It can happen to anyone at anytime.  The other is the inherited form named “Familial” ALS (FALS). Only about 5 to 10% of all ALS patients appear to have FALS. As the disease progresses the symptons become more acute. Paralysis spreads through the body affecting  speech, swallowing, chewing and breathing. Ventilator support is need in late stages

 Pat’s Journey

Pat took the “road less traveled”.  He was a passionate hockey player in Canada. He  concluded in his late teens that he was not at a level to take this road to wealth and fame.

Pat Carr

Pat Carr

06/04–present Associate Professor, Department of Anatomy & Cell Biology, School of Medicine and Health Sciences, University of North Dakota 

1996–98 Research Associate/Adjunct Assistant Professor/Auxilliary Assistant Professor, Department of Anatomy;Wright State University

 07/98–06/04 Assistant Professor, Department of Anatomy & Cell Biology, School of Medicine and Health Sciences, University of North Dakota

Postdoc, National Institutes of Health, Neuroscience, 1994-96

Postdoc, University of Manitoba, Neuroscience, 1992-1994    

Ph.D., University of Manitoba, Physiology, 1992

Next was a stint as an automechanic in Brandon, Canada. The discipline and logic involved in fixing cars catalyzed an interest in Science which led to him going to Brandon University to study Geology. When the oil market collapsed in 1983, he decided to change his studies to Zoology and earned a BS in 1984.

A passion was sparked when he did field research in the Canadien Rockies studying parasites in Columbian Ground  Squirrels. He loved it, but recognized the limited value of continuing thsese studies. This lead to the wide open field of Neuroscience and the opportunity to study and solve problems that could benefit mankind. His graduate work at University of Manitoba and focusing on Neuropathic Pain and the Dorsal Horn. He then moved on to studying Ventral Horn and Motor Control Function for his Post Doc at Wright State.

From Pain to ALS

It was Pat’s work in Pain at the University of North Dakota that brought me into initial contact with him. He generously put some of our key Pain/Inflammation and  Neurotransmission Research Antibodies through their paces. These included some of our Neuropeptide and Neuropeptide Receptors , P2X Receptors and TRPV1s (Vanilloids).

His previous work in studying the Ventral Horn combined with a colleagues mouse model of ALS combined to create a prefect opportunity to advance the understanding of ALS.  Pat cautioned me with this insight:  ”sometimes it is  not what you want to study; it is what you can study.  The model is  SOD1 (superoxide dismutase 1) which is core to FALS.(occurs in only about 10% of the ALS cases).

Pat is broadening the play field by looking at what else is happening in sporadic ALS vs FALS. Specifically, he is looking at modulation of alpha Motor Neurons and how the activity of adjacent Renshaw Cells impact signaling and modulation.  Renshaw Cells act as a “governor” on the activity of these alpha Motor Neurons. 

He is drilling down by studying the signaling of ChAT (Choline Acetyltransferase), VAChT (Vesicular acetylcholine transporter) and related molecules. By gaining a deeper understanding of how Renshaw Cells signaling changes the activity of alpha Motor Neurons in ALS,  Pat and his team are taking steps towards discovering roots causes.

As these root causes are further illuminated, I will be reporting specifics in my blog.

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.

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.