Sunday, May 23, 2010

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-FectTM 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 ( 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 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.

Friday, May 7, 2010

Andy Miller and Enabling RNAi Based Therapies

Deliverying siRNA in vivo is faced with may challenges. Non specific and immune responses are at the top of the list. These hurdles stand in the way of optimally deliverying siRNA in vivo.

That said, I just came across an excellent presentation outlining methods to improve delivery. Creative chemistry indeed! Here's the link:

Andy Miller talking at RNAi Europe 2009
Andy Miller from Imperial College London giving his keynote lecture at RNAi Europe in Berlin. The talk was entitled, 'Enabling RNAi Therapeutics with Safe, Synthetic, Self Assembling Nanoparticles'.
Published : 2009/10/27

Tuesday, May 4, 2010 it Out! 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.