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Off-target Effects (off-target + effects)

Distribution by Scientific Domains

Chemistry	60%
Medical Sciences	20%

Selected Abstracts

Off-Target Effects Related to the Phosphorothioate Modification of Nucleic Acids

CHEMMEDCHEM, Issue 8 2010
Johannes Winkler Dr.
Abstract Phosphorothioate antisense oligonucleotides have been widely used in clinical studies for rational sequence-specific gene silencing. However, several sequence-unspecific off-target effects have been recently described for this compound class. In contrast to siRNA-mediated knockdown of the same gene, the bcl-2 -targeted oblimersen (Genasense, G3139) downregulates a number of proteins involved in apoptotic resistance and several glycolytic enzymes in 607B human melanoma cells. Regardless of their target, phosphorothioate-modified antisense and siRNA compounds, but not oligonucleotides with a phosphodiester backbone, resulted in a similar impact on the proteome. Unspecifically downregulated proteins include cancer markers involved in apoptotic resistance and endoplasmatic reticulum (ER) stress such as the 78,kDa glucose regulated protein (GRP,78), protein disulfide isomerase,A3 (PDIA3, GRP,58), calumenin, and galectin-1, as well as the glycolytic enzymes triose phosphate isomerase, glyceraldehyde phosphodehydrogenase, and phosphoglycerate mutase. The depletion of the glycolytic enzymes is reflected by a decrease in L -lactate production, indicating a partial reversal of the Warburg effect. Compared with other phosphorothioate oligonucleotides, oblimersen generally led to a more pronounced effect both in terms of the number of influenced proteins and the extent of downregulation, suggesting a synergistic effect of Bcl-2 downregulation. [source]

Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacy

FEBS JOURNAL, Issue 22 2009
Hemant K. Kini
To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5, end is less stably hybridized relative to the end containing the passenger strand 5, end. One means by which this is accomplished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5, end. However, there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here, the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand (at the 5, end, nucleotide 1) or the passenger strand (nucleotide 19 from the 5, end) was examined. Subsequently, we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches, which largely agrees with the reduction in eventual silencing efficacy of the siRNAs. Single terminal mismatches led to a small increase in Dicer binding, as expected, but this did not lead to an improvement in silencing activity. These results demonstrate that introduction of mismatches to control siRNA asymmetry may not always improve target silencing, and that care should be taken when designing siRNAs using this technique. [source]

Gene expression silencing with ,specific' small interfering RNA goes beyond specificity , a study of key parameters to take into account in the onset of small interfering RNA off-target effects

FEBS JOURNAL, Issue 11 2008
Sébastien Vankoningsloo
RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments. [source]

Selecting and evaluating native plants for region-specific phytotoxicity testing

INTEGRATED ENVIRONMENTAL ASSESSMENT AND MANAGEMENT, Issue 1 2008
David Olszyke
Abstract In this study, we evaluated methodology to determine risks to terrestrial native plant species from potential herbicide drift, focusing on 1) selection of native species for testing, 2) growth of these species, and 3) variability in herbicide response among native species and compared with crop plants. Native plant species were selected for initial testing on the basis of spatial analysis, which indicated that species from Illinois, USA, were at potential risk for off-target effects of herbicide drift. On the basis of preliminary seed germination tests, 5 native plant species (Andropogon gerardi, Polygonum lapathifolium, Solidago canadensis, Symphyotrichum lateriflorum, and Tridens flavus) were selected for comparison with crops grown in Illinois, normally used in the US Environmental Protection Agency's (USEPA's) Vegetative Vigor Test (Avena sativa, Daucus carota, Glycine max, Solanum lycopersicon, and Zea mays), or both. When treated with low concentrations of a test herbicide, sulfometuron methyl, 2 native species, P. lapathifolium and S. canadensis, were as sensitive as the 5 crop species. The effective herbicide concentrations producing a 25% reduction in shoot dry weight (EC25) for these species, ranged from 0.00015 to 0.0014 times a field application concentration of 52 g/ha active ingredient of sulfometuron methyl. S. lateriflorum and T. flavus were less sensitive than the other native species, whereas A. gerardi was tolerant to sulfometuron methyl with no growth reduction at any herbicide concentration tested. This study indicated that native species can be successfully selected and grown, used in the suite of species used in the USEPA's phytotoxicity test to assess risks of chemical herbicides to nontarget plants. It also showed (with a limited number of species) that native species varied more in sensitivity to simulated herbicide drift than crop species often used in phytotoxicity testing and that a Weibull function was useful to calculate EC25 values when low concentrations of herbicides was used. [source]

RNA interference in pain research

JOURNAL OF NEUROCHEMISTRY, Issue 2 2006
Thomas Röhl
Abstract Within the course of only the last few years, RNA interference (RNAi) has been established as a standard technology for investigation of protein function and target validation. The present review summarizes recent progress made in the application of RNAi in neurosciences with special emphasis on pain research. RNAi is a straightforward method to generate loss-of-function phenotypes for any gene of interest. In mammals, silencing is induced by small interfering RNAs (siRNAs), which have been shown to surpass traditional antisense molecules. Due to its high specificity, RNAi has the potential for subtype selective silencing of even closely related genes. One of the major challenges for in vivo investigations of RNAi remains efficient delivery of siRNA molecules to the relevant tissues and cells, particularly to the central nervous system. Various examples will be given to demonstrate that intrathecal application of siRNAs is a suitable approach to analyse the function of receptors or other proteins that are hypothesized to play an important role in pain signalling. Intensive efforts are currently ongoing to solve remaining problems such as the risk of off-target effects, the stability of siRNA molecules and their efficient delivery to the CNS. RNAi has thus demonstrated that it is an extremely valuable tool for the development of new analgesic drugs. [source]

Review article: RNA interference , potential therapeutic applications for the gastroenterologist

ALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 9 2008
R. S. PELLISH
Summary Background, A new technique of gene regulation, termed RNA interference, has emerged recently. RNA interference utilizes short double-stranded RNA to inhibit selectively gene expression of complementary RNA nucleotide sequences after transcription, but prior to translation. Gastrointestinal and hepatic disorders may be particularly amenable to therapeutic RNA interference intervention because of the relative ease of delivery of drugs to the gastrointestinal tract and liver. Aim, To examine the published literature for potential clinical uses of RNA interference in gastroenterology and speculate on future therapies for luminal disease. Methods, Reports were identified using PubMed and the search term ,RNA interference', focusing on therapeutic uses related to gastrointestinal and liver disease. Results, Cellular and animal models demonstrate the potential application of short-interfering RNA-based therapies for viral hepatitis and inflammatory bowel disease. With validation of specific targets and better in vivo delivery of short-interfering RNA, RNA interference may represent a new frontier for molecular-targeted therapy in gastroenterology and hepatology. Conclusions, Short-interfering RNA provides a novel and specific means to inhibit gene expression. Translation to the clinical arena will require further definition of side-effects, off-target effects and delivery systems. Ultimately, mucosally applied or endoscopically delivered short-interfering RNA could be one of the earliest clinical uses of short-interfering RNA therapy. [source]

Inhibition of myostatin with emphasis on follistatin as a therapy for muscle disease

MUSCLE AND NERVE, Issue 3 2009
Louise R. Rodino-Klapac PhD
Abstract In most cases, pharmacologic strategies to treat genetic muscle disorders and certain acquired disorders, such as sporadic inclusion body myositis, have produced modest clinical benefits. In these conditions, inhibition of the myostatin pathway represents an alternative strategy to improve functional outcomes. Preclinical data that support this approach clearly demonstrate the potential for blocking the myostatin pathway. Follistatin has emerged as a powerful antagonist of myostatin that can increase muscle mass and strength. Follistatin was first isolated from the ovary and is known to suppress follicle-stimulating hormone. This raises concerns for potential adverse effects on the hypothalamic,pituitary,gonadal axis and possible reproductive capabilities. In this review we demonstrate a strategy to bypass off-target effects using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno-associated virus (AAV) to muscle. The transgene product is a peptide of 315 amino acids that is secreted from the muscle and circulates in the serum, thus avoiding cell-surface binding sites. Using this approach our translational studies show increased muscle size and strength in species ranging from mice to monkeys. Adverse effects are avoided, and no organ system pathology or change in reproductive capabilities has been seen. These findings provide the impetus to move toward gene therapy clinical trials with delivery of AAV-FS344 to increase size and function of muscle in patients with neuromuscular disease. Muscle Nerve 39: 283,296, 2009 [source]

Off-Target Decoding of a Multitarget Kinase Inhibitor by Chemical Proteomics

CHEMBIOCHEM, Issue 7 2009
Enrico Missner
Abstract Unbiased: Chemical proteomics was used to profile compound interactions in an unbiased fashion. We present here the application of different compound-immobilization routes for decoding nonprotein kinase off-targets of the multitarget kinase inhibitor C1, which interacts with distinct compound moieties. Since the approval of the first selective tyrosine kinase inhibitor, imatinib, various drugs have been developed to target protein kinases. However, due to a high degree of structural conservation of the ATP binding site, off-target effects have been reported for several drugs. Here, we report on off-target decoding for a multitarget protein kinase inhibitor by chemical proteomics, by focusing on interactions with nonprotein kinases. We tested two different routes for the immobilization of the inhibitor on a carrier matrix, and thus identified off-targets that interact with distinct compound moieties. Besides several of the kinases known to bind to the compound, the pyridoxal kinase (PDXK), which has been described to interact with the CDK inhibitor (R)-roscovitine, was captured. The PDXK,inhibitor interaction was shown to occur at the substrate binding site rather than at the ATP binding site. In addition, carbonic anhydrase 2 (CA2) binding was demonstrated, and the determination of the IC50 revealed an enzyme inhibition in the submicromolar range. The data demonstrate that different compound immobilization routes for chemical proteomics approaches are a valuable method to improve the knowledge about the off-target profile of a compound. [source]

Off-Target Effects Related to the Phosphorothioate Modification of Nucleic Acids

Exploring Chemical Modifications for siRNA Therapeutics: A Structural and Functional Outlook

CHEMMEDCHEM, Issue 3 2010
Siddharth Shukla
Abstract RNA interference (RNAi) is a post-transcriptional gene silencing mechanism induced by small interfering RNAs (siRNAs) and micro-RNAs (miRNAs), and has proved to be one of the most important scientific discoveries made in the last century. The robustness of RNAi has opened up new avenues in the development of siRNAs as therapeutic agents against various diseases including cancer and HIV. However, there had remained a lack of a clear mechanistic understanding of messenger RNA (mRNA) cleavage mediated by Argonaute2 of the RNA-induced silencing complex (RISC), due to inadequate structural data. The X-ray crystal structures of the Argonaute (Ago),DNA,RNA complexes reported recently have proven to be a breakthrough in this field, and the structural details can provide guidelines for the design of the next generation of siRNA therapeutics. To harness siRNAs as therapeutic agents, the prudent use of various chemical modifications is warranted to enhance nuclease resistance, prevent immune activation, decrease off-target effects, and to improve pharmacokinetic and pharmacodynamic properties. The focus of this review is to interpret the tolerance of various chemical modifications employed in siRNAs toward RNAi by taking into account the crystal structures and biochemical studies of Ago,RNA complexes. Moreover, the challenges and recent progress in imparting druglike properties to siRNAs along with their delivery strategies are discussed. [source]