siRNA Delivery Group on Linkedin

I wanted to make readers aware of an excellent discussion group on Linkedin named "siRNA Delivery". Included are tip, updates on commercialization and key publications.

Here're some examples:

• Alnylam Announces Publication of Pre-clinical Results with ALN-HTT, an RNAi Therapeutic for the Treatment of Huntington’s Disease, in Experimental Neurology
• Delivering siRNA to Neurons
• Life Technologies develops new drug delivery technology
• Recent experiments demonstrate a novel method of liposomal encapsulation of siRNA/RNA/DNA at 4C, could be a promising method to minimize degradation.

Happy reading.

β-arrestin siRNA Delivery in vivo and Increased Analgesia

I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of Cav1.2, DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.

I would like to congratulate Dr. C.R. Lin and his team at National Taiwan University College of Medicine for silencing β-arrestin expression in vivo and the impact on opioid based analgesia. The results could be good news for improving opioid based pain therapies: C.-H.Yang, H.-W. Huang, K.-H.Chen, Y.-S.Chen, S.-M.Sheen-Chen and C.-R.Lin. Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats. Br. J. Anaesth. (2011) doi: 10.1093/bja/aer291.

Background: Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal.

Methods: Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h−1) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg−1) was performed to assess morphine dependence.
Results: In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose–response curve, as measured by per cent maximal possible effect (MPE), such that the AD50 was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal.
Conclusions: We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.

intra-i-Fect and intravenous delivery of siRNA

Deliver siRNA in-vivo with stunning results! Introductory Special-200 to 600 USD (valid through 9/30/2010)
These intra-i-Fect kits are designed to deliver siRNA in vivo via intravenous injections with high efficiency to specific tissue in rats and mice. The protocol involves these simple steps: prep, mix, dry, hydrate and inject.

Figure: siRNAs knock down profiles of the gene related to cancer, diabetes, obesity, steatosis hepatitis, cirrhosis and a gene specifically expressed in endothelial cells in liver

They are developed using a proprietary platform that uses nano-particles as the delivery vehicle. This platform enables:
•Effective delivery (60%+ knockdown) with no toxicity.
•Scalable to high throughput siRNA based gene screening.
•Consistent and reproducible results.

Raf-1- selective siRNA and Response to Pain

I've reported Researchers' success in knocking down in vivo DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more using Neuromics' i-Fect^TM siRNA transfection reagent .

I am pleased to add Raf-1 to this growing list. Here's a recent publication by Dr. EV Varga, University of Arizona:

S Tumati, WR Roeske, T Largent-Milnes, R Wang, TW Vanderah and EV Varga. Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1- selective siRNA. This is an Accepted Article that has been peer-reviewed and approved for publication in the British Journal of Pharmacology, but has yet to undergo copy-editing and proof correction. Please cite this article as an "Accepted Article"; doi: 10.1111/j.1476-5381.2010.00869.x.

Background and purpose: Long-term morphine treatment enhances pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats.

Experimental approach: Rats were intrathecally (i.th) injected with a Raf-1- selective small interfering RNA (siRNA) mixture for 3 days, and were subsequently infused with saline or morphine, s.c. for seven days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using ELISA and immunohistochemistry.

Key results: Selective knockdown of spinal Raf-1 protein levels by i.th Raf-1- selective siRNA pre-treatment significantly attenuated sustained morphine-mediated upregulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance.

Conclusions and implications: Raf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain.

RNAi.net-Check it Out!

RNAi.net is a portal that has done an excellent job at providing a gateway to many resources to help researchers using siRNAs for gene expression analysis.

There webcast link is particulary useful. Included is a presentation by one of our collaborators: Dr. Mark Behlke. Here's the abstract:

Dicer-substrate siRNAs (DsiRNAs) are synthetic oligonucleotides that are processed by Dicer prior to RISC loading. DsiRNAs often show improved potency over traditional siRNAs in vitro and can have similar benefits in vivo. In collaboration with Dicerna Pharmaceuticals, systematic high throughput screening of DsiRNAs is in progress to identify ultra-potent sites in pharmaceutically relevant target genes. The results of a KRAS screening project will be discussed where over 400 synthetic siRNAs were tested in human and mouse cells. Chemical modification patterns have been defined that improve nuclease stability of the DsiRNA while retaining high potency and evade detection by the innate immune system. These improvements to DsiRNA design will be presented, which have particular utility for in vivo applications. In addition to work in RNAi, results will be presented relating to a new gene-knockdown technology that uses synthetic adaptor oligonucleotides to recruit the nuclear U1 snRNP complex to cleave nascent mRNAs prior to polyadenylation. RNAi and U1 adaptors work by different mechanisms at distinct sub-cellular locations and can be used together to improve knockdown of difficult targets.