DsiRNA Transfection Alliance

Neuromics has formed an alliance to partner with IDT in forwarding capabilties to deliver RNAi to Glia, Neurons and the Nervous System using i-Fect and 27mer Dicer Substrate siRNA (DsiRNA).

 

Featured DsiRNA Publication:

 

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); doi:10.1038/mt.2008.98

"In this study, we demonstrate the efficacy of 27-mer DsiRNA in reducing the expression of a specific G-protein coupled-receptor (GPCR) gene in rat spinal cord and DRG. Low doses of DsiRNA formulated in i-Fect, when administered by IT injection, induced a sustained reduction in the neurotensin receptor-2 (NTS2) GPCR mRNA and protein levels for 3–4 days. The reduction in NTS2 resulted in the expected behavioral changes in nociception. No apparent toxicity or nonspecific side effects were exhibited during the study period, and our results overall highlight the feasibility of using DsiRNA in pain research.”

Comments by Dr. Mark Behlke, CSO, IDT

 

Dicer-substrate siRNAs (DsiRNAs) have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration. “IDT got into RNAi research in collaboration with John Rossi at The City of Hope and the Beckman Research Institute five years ago,” explained Dr. Behlke. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RISC and serve as a sequence-specific guide to target degradation of complementary mRNA species.
Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3´-overhangs on the termini, reported Dr. Behlke. These duplexes are transfected into cells lines, directly mimicking the products made by dicer in vivo. Most siRNA sequences can be administered to cultured cells or to animals without eliciting an interferon response.

“We observed,” added Dr. Behlke, “that the use of slightly longer sequences that were substrates for dicer showed improved potency, which we theorize relates to participation of dicer in RISC loading. We are now focusing on the use of these compounds in vivo.”

 

IDT recently completed a collaborative study with the laboratory of professor Phillipe Sarret at the Université de Sherbrooke in Quebec. The collaboration studied the use of DsiRNA to knockdown the GPCR NTS2 (neurotensin type 2 receptor) in rat spinal cord and dorsal root ganglia. The RNA duplexes were administered by intrathecal injection in a cationic lipid slurry. Stimulation of NTS2 with a chemical agonist resulted in analgesia. Pain responses were monitored in treated animals by dipping their tails in hot water with and without the chemical agonist.

“The anti-NTS2 DsiRNA treated animals showed a marked difference of response to the test stimulus,” said Dr. Behlke. “We recorded differences of up to five seconds, which is quite a long time for a rat to sit with its tail in hot water. While interesting, this work mainly represents a pilot study to demonstrate the feasibility of using DsiRNA to study pain pathways in rats. We were amazed at the low dose it takes to get knockdown—we used 1 mcg/200 g rat, which is only a 0.005 mg/kg dose.” Modulating CNS disease and affecting brain processes is clearly possible, but better methods of delivery are going to be needed to move this approach from a research tool into the clinic, noted Dr. Behlke

 

We currently have several projects underway investigating the potency of 27 vs 21mers both in vitro and in vivo. We will be publishing results here and to our blog. Here are links to key references:

 

 

Other in vivo Publications:

 

X.-W. Dong, S. Goregoaker, H. Engler, X. Zhou, L. Mark, J. Crona, R. Terry, J. Hunter and T. Priestley. Small interfering RNA-mediated selective knockdown of Na_V1.8 tetrodotoxin-resistant sodium channel reverses mechanical allodynia in neuropathic rats. Neuroscience Volume 146, Issue 2, 11 May 2007, Pages 812-821

For in vivo studies, siRNA stock solution (1 mg/ml) was prepared using siRNA suspension buffer (Qiagen). The solution was then heated to 90 °C for 1 min followed by 60 min incubation at 37 °C. Aliquots were stored at -20 °C. On the day of the injection, an aliquot of the siRNA was thawed on ice and then mixed with the transfection reagent, iFect (Neuromics Antibodies, Northfield, MN, USA), to yield a final concentration of 0.2 ug/ ul. A total of 100 ul of the siRNA solution or vehicle (20 ul of suspension buffer in 80 ul of iFect; 1:4 v/v) was locally delivered to lumbar DRGs through implanted epidural catheter by slow injection. The catheter was flushed with 15 ul sterile saline

 

Luo MC, Zhang DQ, Ma SW, Huang YY, Shuster SJ, Porreca F, Lai J (2005). An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons. Molecular Pain 2005, 1:29.

 

Priti Kumar, Sang Kyung Lee, Premlata Shankar, N. Manjunath (2006) A Single siRNA Suppresses Fatal Encephalitis Induced by Two Different Flaviviruses. PLOS Medicine,  Volume 3 | Issue 4 | APRIL 2006