RNAi Researchers Galvanized by Advances

Technology's Viability in Drug Development Is Finally Established
Author: Elizabeth Lipp
Publication: Genetic Engineering & biotechnology News
Publisher: Mary Ann Liebert, Inc. publishers
Date: Jun 1, 2008
Copyright © 2008 GEN Publishing

Article Link: http://www.genengnews.com/articles/chitem.aspx?aid=2493

Notable Quotables:

“Long dsRNAs have been employed for many years as a means to modulate gene expression in plants, yeast, and C. elegans,” noted Mark Behlke, M.D., Ph.D., svp of molecular genetics and CSO at Integrated DNA Technologies (IDT; www.idtdna.com).
“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.”

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Central Delivery of DsiRNA

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.

Images: Cellular uptake of Texas Red–tagged Dicer-substrate small-interfering RNA (DsiRNA) by spinal nociceptive structures. (a,b) Distribution of fluorescence in lumbar dorsal root ganglia at 24 hours after intrathecal injection of a control siRNA conjugated with Texas Red (1 μg administered twice with a 24-hour interval; n = 3). As seen by confocal microscopy, the staining is not uniformly distributed among the cells. Higher-magnification images also show that the fluorescent signal is detected in the form of small intracytoplasmic hot spots, sparing the nucleus. (c,d) Expression of Texas Red–tagged DsiRNA in a dorsal spinal cord section taken from an L5 segment. Fluorescence clusters are present in the cytoplasm of the cells. Note that the labeling is also detected in neuronal processes. Scale bar: 60 μm in a, 30 μm in b,25 μm in c and 15 μm in d. Courtesy of Dr. Nicolas Beudeat. Published in Molecular Therapy (2008); doi:10.1038/mt.2008.98

Use of Dicer Substrate siRNAs

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.