Dealing with Interferon Response When Doing RNAi

From AlleleBlog: http://allelebiotech.com/blogs/2010/09/dealing-with-interferon-response-when-doing-rnai/

Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.

Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.

Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.

Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.

Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.

To read the wholr blog, click here.

MicroRNA-132 is identified as a stimulator of neovascularization

http://www.allelebiotech.com/News//index.php?mod=article&cat=RNAi&article=774

By looking for miRNAs that are upregulated by VEGF or bFGF in umbilical vein endothelial cells (HUVECs) AND an ES neovascularization model, researchers in the Cheresh group at UCSD and Moores Cancer Center and the King lab at U Michigan identified miRNA-132 as a critical switch for activating endothelial cells during angiogenesis. The result, published in Aug 1st’s Nature Medicine, is most significant in that miRNA-132 could become a target for tumor prevention given the relevance of pathological blood vessel formation to tumors.


Profiling of miRNA during pathological development has been fruitful in many cases, including one conducted by Allele Biotech’s researchers and colleagues from Nanjing Medical University where a number of miRNAs were identified with dramatic changes that are specific to several stages of adipogenesis. It is somewhat surprising that miRNA profiling has not been done in more development models or in more systematic ways. In fact, other miRNAs have been identified as stimulators of angiogenesis previously, such as miR-296, which, different from miRNA-132, is “upregulated only in growth factor-treated endothelial cells but not in the developmental angiogenesis screen using human embryonic stem cells.”

The mechanism by which miRNA-132 enhances blood vessel formation has been pinpointed to a RAS regulator. The method used for predicting miRNA targets was by using 3 algorithms for finding multiple sites in the 3’-UTR. The technique for analyzing miRNA-12 effects was mostly based on using an anti-miRNA-132 oligo. Both cell and animal models were used in this study. A type of tumor-specific integrin-targeted nanoparticle was used to deliver anti-miRNA-132 to tumor sites in mice.

Anand et al. Nature Medicine, 08/01/2010
http://www.nature.com/nm/journal/vaop/ncurrent/full/nm.2186.html

Allele Granted Broad Patent on RNAi in China

Allele Biotechnology & Pharmaceuticals, a San Diego based private company with associate offices and laboratories in China and distribution channels in 30 countries, was granted a major landmark patent in China in the field of RNA interference (RNAi). The patent CN02828345.7, issued on January 20, 2010, covers compositions of DNA molecules that can be transcribed into RNAi-mediating RNA molecules, including the commonly used shRNA and miRNA-like designs. The patent also grants Allele Biotech rights to the process of introducing such DNA molecules into cells. To induce gene silencing by RNA interference, researchers often bring DNA molecules that encode interfering RNAs into cells via plasmid or viral vectors. The rights to use related technologies for the purposes of completely or partially abolishing gene functions through the mechanism of RNAi are granted to Allele Biotech.



Additional claims include methods of studying gene functions using DNA-encoded RNAi agents, or modifying gene expression profile by introducing gene expression-altering DNA molecules that will induce RNAi. The patent further protects the use of DNA-mediated RNAi in creating cell, animal models, and for curing human diseases. Allele Biotech intends to fully realize the value of this broad patent by providing opportunities to R&D centers, service providers, and reagent sellers to license at reasonable fees, so that this great technology will continue to be widely used and further developed through original research and investment.



For more views and comments by Allele Biotech and responses from others, please visit the Allele Blog on the same topic.



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For further information: 858-587-6645, Fax 858-587-6692 RNAi@allelebiotech.com

Choosing siRNA, shRNA, and miRNA for Gene Silencing

http://allelebiotech.com/blogs/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/
...shRNA can be introduced by DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format such as Allele’s LineSilence platform, requires creating DNA constructs and sequence verification; a taxing work load if multiple genes need to be studied. However, once the constructs are made, they can be reproduced easily and inexpensively. It is difficult to directly compare the effectiveness of siRNA versus shRNA on a per molecule basis because RNA polymerase III (Pol III) promoters such as U6 or H1 commonly used to express shRNAs can make thousands of copies of shRNA from a single DNA template. However when both siRNA and shRNA are produced the same way, e.g. synthesized chemically, shRNA is reported to be somewhat more effective [6, 7]. For the goals of this research, the most important advantage using shRNA can provide over siRNA is that it can be carried on a lentiviral vector and introduced into a wide variety of cells.

Similar to the comparison between siRNA versus shRNA, it is also difficult to rank the efficiency of shRNA versus miRNA from published data, partly due to different results from different experimental systems. There have been several reports that showed shRNA can cause significant cell toxicity, especially in vivo such as after injection into mouse brain. It was originally reasoned that highly efficient expression from Pol III promoters might overwhelm the cellular machinery that is needed to execute endogenous RNAi functions such as transporting miRNA from the nucleus to the cytoplasm. It was later found out that even using Pol III promoter to create miRNA could still mitigate the toxic effects of shRNA [8]. Since shRNA and miRNA are processed by endonuclease Dicer before being incorporated into RNA induced silencing complex (RISC), the exact identity of siRNAs produced from a given shRNA or miRNA targeting the same region on the mRNA are not known in most of the earlier studies. By designing shRNA and miRNA to give exactly the same processed siRNAs, Boudreau et al. showed that shRNA is actually more potent than miRNA in various systems [9].

New Product/Service of the Week (02-01-10 to 02-07-10): Lentrivirus retrovirus shRNA Packaging Services as low as under $900 per virus.

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For References see original post here please.