Spinal Cord Injury and Ependymal Cells

This research sheds light on the natural regeneration that occurs in the area of damaged spinal cord tissue. The surprise here is the role that Edendymal Progenitor Cells are playing in repair mechanism vs Neural Stem Progenitors. 

In this publication, researchers have employed genetic fate mapping to characterize a candidate neural stem cell population in the adult spinal cord and show that close to all in vitro neural stem cell potential resides within the population of ependymal cells. Ependymal cells give rise to a substantial proportion of scar-forming astrocytes as well as to some myelinating oligodendrocytes after spinal cord injury. Modulating the fate of ependymal cell progeny after injury could potentially promote the generation of cell types that may facilitate recovery after spinal cord injury.

Meletis K, Barnabé-Heider F, Carlén M, Evergren E, Tomilin N, et al. (2008) Spinal Cord Injury Reveals Multilineage Differentiation of Ependymal Cells . PLoS Biol 6(7): e182 doi:10.1371/journal.pbio.0060182

Dr. Steve Stice Podcast

Inteview with Steve Stice.

Podcast by ClearCast

This Podcast gives is a good primer on Embryonic Stem Cells (ESCs)…where they come from, how they are used and the promise they have for helping researchers cure human diseases.

The listener will gain insight how ECSs when manipulated into mature neuronal cell lines can accelerate the pace of neurological research for scientists working on treatments for spinal cord injuries and neurological diseases such as Parkinson’s, Huntington’s, Alzheimer’s, ALS and possibly even depression.

On Deck-Dr. Steve Stice

We read about the promise of stem cells in the news every day. They could prove to be ”magic bullets” for curing diseases like Alzheimer’s. Parkinson’s, MS and others. Stem Cell Research is also surrounded with controversy as currently cells are often harvested from human embryos and fetuses.I believe top researchers will prove to be the voice of reason in the human stem cell debate as they are the ones best positioned to know the risks, limitations and potential.  

For our August Profile, I am honored to be featuring Dr. Steve Stice. I have had the pleasure of working with Dr. Stice both in his role as Professor and Director of the Regenerative Bioscience Center and Research Alliance Eminent Scholar endowed Chair at the University of Georgia and as Founder and Chief Scientific Officer at Aruna Biomedical.

He has over 16 years of research and development experience in biotechnology and is a co-founder of five biotechnology companies.  He was named one of the 100 Most Influential Georgians by Georgia Trend magazine.  He produced the first cloned rabbit in 1987 and the first cloned transgenic calves in 1998 (George and Charlie).  In 1997 his group produced the first genetically modified embryonic stem cell derived pigs and cattle.  This research led to publications in Science and Nature journals, national news coverage (CBS, NBC, ABC and CNN) and the first US patents on cloning animals and cattle embryonic stem cells.  In 2001, Dr. Stice announced the first cloned animal (calf) from an animal that was dead for 48 hours.  In 2005, his stem cell group published the first work on deriving motor neurons from stem cells.  Motor neurons are damaged lost during the progression of several diseases such as ALS and spinal muscular atrophy.  Throughout his career he has published and lectured on cloning and stem cell technologies.  Prior to joining the University of Georgia, Dr. Stice was a co-founder and Chief Scientific Officer at Advanced Cell Technology, a company developing cloning and stem cell technology.

Here is What is  Currently Hot in the Stice Lab:
New neural stem cells technology developed in my lab was transferred to a commercial entity, Aruna biomedical. This is the first commercialized product derived from human embryonic stem cell using federally approved stem cell lines.

  • We have produced neurons that have neural functions
  • We are working with the Navy to use our neural cells as biosensors for environmental toxins 
  • We have vascular stem cells that have characteristics that may make them suitable for  transplantation
  • We collaborate with a new company call Aruna BioMedical  that will stem cells for neural research and drug discovery
  • Developed a method to test new compounds for Alzheimer’s disease using our neural stem cell
  • We are one of five NIH stem cell training centers and have taught Scientists from Georgia to Bombay India new stem cell techniques
  • In Georgia, we produced over 50 cloned calves and 100 cloned pigs.
  • We were also the first to produce a clone from an animal that had been dead for 48 hours. This opens new opportunities in agriculture and preserving endangered species. 

Improving 27mer DsiRNA Preformance

The DsiRNA story marches forward with yet another important publication. We will give you an opportunity to upload the fulltext article at the end of this posting.

Dr. Mark Behlke, Dr John Rossi and team have been gaining deeper understanding of the Mechanism of Dicer-substrate small-interfering RNA (DsiRNA) processing. This understanding is leading to better and better designs of the RNA duplexes. These designs or chemical modifications are necessary steps in the drug design and development process.

This publication looks at design from the perspective of:

  • Nuclease Stability
  • Pharmacokinectics
  • immune response

I believe this is an important publication for researchers wanting to better understand:

  1.  The mechanisms behind successful delivery of DsiRNA for gene expression studies.
  2. Variations in potency.

upload article:  oligo-18-p187-2008-collingwood-dsirna-modifications1

 

More Good News for DisRNA Research

As a follow on to the potential of 27mer DsiRNAs as a key platform for drug discovery and development, we are happy to report:

15.07.2008 13:34

Dicerna Pharmaceuticals, a Second Generation RNA Interference Company, Completes Second Closing of Series A Financing; Unveils Strategic Focus

Dicerna Pharmaceuticals, Inc. (www.dicerna.com), an RNA interference company developing novel therapeutics utilizing proprietary Dicer Substrate Technology™, today announced a second closing of its Series A financing, receiving a total of $8.4 million in additional venture capital from new investor Abingworth and existing investors Oxford Bioscience Partners and Skyline Ventures. With this additional capital, Dicerna has completed a $21.4 million Series A financing round. Pursuant to this second closing, Vincent J. Miles, Ph.D., venture partner at Abingworth has joined Dicerna’s board of directors. Prior to Abingworth, Dr. Miles was senior vice president of business development at Alnylam Pharmaceuticals, an Abingworth portfolio company. Read entire press release.

We will continue to keep you updated on the Dicerna Story.

 

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.

Dr. Behlke’s 27mer DsiRNA Story Coming Soon

Learn the News Behind the News on 27mer DsiRNA. Includes the story of the how the potentcy of 27mer siRNA was discovered and exiting developments regarding the understanding of the role of the Dicer Complex and RISC. Also featured will be advances in 27mer oligo design and how the technology is being used in basic research and drug discovery.

Here’s a publication highlighting the roots of the story:

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