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.

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: http://www.labtube.tv/avc-interest.aspx?i=5&c=1&v=327.

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

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.

Direct Application of siRNA for In Vivo Pain Research

My friends at McGill University have recently published in depth methods for using siRNA to study pain. Dr. Philippe Sarret have done extensive work delivering siRNA + i-Fect^TM in vivo for gene expression analysis of specific pain receptors.

Here's a link to the book chapter from Springer Protocols:

25. Direct Application of siRNA for In Vivo Pain Research
By: Philippe Sarret , Louis Doré-Savard, Nicolas Beaudet
Affiliation(s): (1) Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
Book Title: RNA Interference: From Biology to Clinical Applications
Series: Methods in Molecular Biology Volume: 623 Pub. Date: May-01-2010 Page Range: 383-395 DOI: 10.1007/978-1-60761-588-0_25

Abstract: Pain is the new burden of the twenty-first century, raising enormous socio-economic costs to developed and underdeveloped countries. Chronic pain is a central nervous system (CNS) pathology, affecting a large proportion of the population. Morphine and its derivatives are still the golden clinical standards for treating pain although they induce severe side effects. To this day, we still have poor understanding of nociceptive pain and its underlying complex mechanisms; furthermore, novelty in clinical analgesics is lacking.

RNA interference technologies are promising both for pain research and treatment. This genetic approach will likely provide new insights into pain mechanisms and eventually offer nonpharmacological therapeutic approaches. In vivo research is thus crucial to reach this goal. Preclinical studies on rodents are necessary to validate small interfering RNA (siRNA) candidates and to target precise physiological pain modulators. Aiming treatment at the CNS is delicate work, and here we will describe how to perform adequate pain research using siRNA, including siRNA preparation and injection, animal behavioral models, and CNS tissue collection.

i-Fect and More Knockdown Success

Neuromics' Customers have published their success using i-Fect^TM for gene expression studies Genes studied include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.

We are now pleased to add knockdown L Calcium Channel Subtypes to study differential effects of neuropathic pain.

In this study researchers showed specific knockdown of CaV1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-CaV1.2 siRNAs confirmed the preceding results.

Here's a link to the related pub: Pascal Fossat, Eric Dobremez, Rabia Bouali-Benazzouz, Alexandre Favereaux, Sandrine S. Bertrand, Kalle Kilk, Claire Léger, Jean-René Cazalets, Ülo Langel, Marc Landry and Frédéric Nagy. Knockdown of L Calcium Channel Subtypes: Differential Effects in Neuropathic Pain. The Journal of Neuroscience, January 20, 2010, 30(3):1073-1085; doi:10.1523/JNEUROSCI.3145-09.2010

We used siRNA targeting several splice variants of CaV1.2 ("Silencer Select Pre-designed and Validated siRNA", Ambion). They consisted of a pool of two 21 nt duplex. siRNAs were selected to target two distinct CaV1.2 mRNA regions to enhance silencing. The antisense sequences were as follows: UCUAUUGUCAUAUCGCAGG and UAUCCGAACAGGUAUAGAG.

In contrast to PNA, these siRNAs targeted the 5'-coding region. Mismatch siRNA was a nontargeting 21 nt duplex designed as a negative control. The siRNAs (2 µg) were solubilized in 10 µl of reagent i-Fect (Neuromics) following Neuromics instructions and published protocol (Luo et al., 2005), and applied intrathecally according to the same protocol as for the PNA.

Delivering siRNA in Mice for Studying Opioid-Induced Hyperalgesia

Researchers have successfully delivered siRNA in-vitro and in-vivo using Neuromics' i-Fect ™ siRNA Transfection Reagent. Gene expression studies include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.

Here's a link to all transfection publications: Transfection Kit Pubs

We are pleased to present yet another study and related publication. This includes one of the first successful delivery of siRNA in mice using i-Fect ™ :

Yan Chen, Cheng Yang, and Zaijie Jim Wang. Ca2+/Calmodulin-Dependent Protein Kinase II Is Required for the Initiation and Maintenance of Opioid-Induced Hyperalgesia. The Journal of Neuroscience, January 6, 2010, 30(1):38-46; doi:10.1523/JNEUROSCI.4346-09.2010.

...KN93 and KN92 were administered intrathecally by percutaneous puncture through the L5-L6 intervertebral space, as described previously (Hylden and Wilcox, 1980; Chen et al., 2009). A lateral tail flick was considered as success of the intrathecal injection. To inhibit CaMKII, CaMKII was targeted by small interfering RNA (siRNA). Four days after morphine pellet implantation, mice were treated with CaMKII siRNA (5'-CACCACCAUUGAGGACGAAdTdT-3', 3'-dTdTGUGGUGGUAACUCCUGCUU-5') (Zayzafoon et al., 2005) or Stealth RNAi negative control (Invitrogen) (2 µg, i.t., twice per day for 3 consecutive days). These oligos were mixed with the transfection reagent i-Fect (Neuromics), in a ratio of 1:5 (w/v) (Luo et al., 2005). Mechanical and thermal sensitivity tests were performed daily...

Using i-Fect for treatment of Glioblastomas

Dr. Swapan K. Ray, Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine and his team should positive results in reducing growth of Glioblastomas by knocking down hTERT expression using Neuromics' i-Fect ™ siRNA Transfection Kit. Here's the related pub:

Joseph George, Naren L. Banik, Swapan K. Ray. Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma. Clin Cancer Res 2009;15(23):7186–95

...with i-Fect transfection reagent (Neuromics) to obtain 5 μg DNA/10 μL of injection volume...

Results: In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-γ. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression.

Conclusions: Our study showed that the combination of hTERT siRNA and IFN-γ effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-γ is a promising therapeutic strategy for controlling the growth of human glioblastoma.