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Chabot S.,CNRS Institute of Pharmacology and Structural Biology | Chabot S.,Toulouse 1 University Capitole | Orio J.,CNRS Institute of Pharmacology and Structural Biology | Orio J.,Toulouse 1 University Capitole | And 9 more authors.
Molecular Therapy

Micro-RNAs (miRNAs) are small regulatory RNAs that play an important role in disease development and progression and therefore represent a potential new class of therapeutic targets. However, an effective and safe clinical approach for miRNA inhibition remains elusive, primarily due to the lack of effective delivery methods. We proposed to inhibit miRNA by electrotransferring an antisense DNA oligomer containing locked nucleic acids (LNAs) (LNA/DNA oligomer). We observed that electropulsation (EP) led to a strong cellular uptake of LNA/DNA oligomer. The LNA/DNA oligomer electrotransfer mechanism and intracellular localization were visually investigated in real time at the single-cell level. Cyanine 5-labeled oligonucleotide entered exclusively during pulse application on the side of the permeabilized cell membrane facing the cathode, driven by electrophoretic forces. Minutes after the electrotransfer, the LNA/DNA oligomer diffused into the nucleus. EP provided the anti-miRNA oligomer with immediate and direct access to its cytoplasmic mature miRNA target and/or its nuclear precursor miRNA target. We then demonstrated using a LNA/DNA oligomer anti-miR34a that LNA/DNA oligomer electrotransfer decreased the level of the miR34a target and induced its functional inhibition. Our findings show that using the electrotransfer technique for LNA-based oligonucleotide delivery is a promising therapeutic strategy to silence deleterious miRNAs overexpressed in diseases. © The American Society of Gene & Cell Therapy. Source

Orio J.,CNRS Institute of Pharmacology and Structural Biology | Orio J.,Toulouse 1 University Capitole | Bellard E.,CNRS Institute of Pharmacology and Structural Biology | Bellard E.,Toulouse 1 University Capitole | And 12 more authors.
Journal of RNAi and Gene Silencing

Low biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide: The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/ DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFPRab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets. © The Author(s): First Published by Library Publishing Media. Source

Jacobsen N.,Exiqon
Methods in molecular biology (Clifton, N.J.)

This chapter describes a method for the isolation of intact polyadenylated mRNA using LNA oligo(T) capture. The method enables efficient isolation of poly(A)(+) RNA directly from guanidinium thiocyanate (GuSCN)-containing cell or tissue extract by combining the design of biotinylated LNA oligo(T) capture probes with subsequent immobilization of the captured poly(A)(+) RNA onto streptavidin-coated magnetic particles. In contrast to DNA oligo-dT and polyT PNA based mRNA isolation techniques, the LNA oligo(T) capture method allows poly(A) selection in the presence of 4 M GuSCN cell lysis buffer, which is needed for efficient inactivation of endogenous RNases. In addition, LNA oligo(T) facilitates highly efficient poly(A)(+) isolation at elevated temperatures compared to standard oligo(dT) technology. The successful use of the LNA oligo(T) capture method in recovery of mRNA from human cells and the subsequent use of the mRNA in northern blotting analysis, RT-PCR and qRT-PCR are demonstrated. Source

Mestdagh P.,Ghent University | Hartmann N.,Novartis | Baeriswyl L.,Novartis | Andreasen D.,Exiqon | And 32 more authors.
Nature Methods

micrornAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. several measurement platforms have been developed to determine relative mirnA abundance in biological samples using different technologies such as small rnA sequencing, reverse transcription- quantitative PCr (rt-qPCr) and (microarray) hybridization. in this study, we systematically compared 12 commercially available platforms for analysis of micrornA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference rnA, human brain rnA and titrations thereof, human serum samples and synthetic spikes from micrornA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specifcity and concordance of differential expression. the results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative micrornA gene expression platform for particular study goals. © 2014 Nature America, Inc. All rights reserved. Source

Husted S.,Copenhagen University | Sokilde R.,Exiqon | Rask L.,Copenhagen University | Cirera S.,Copenhagen University | And 3 more authors.
Molecular Pharmaceutics

Multidrug resistance (MDR) poses a major obstacle to successful chemotherapeutic treatment of cancer, and often involves multiple genes, which may be regulated post-transcriptionally by microRNAs (miRNAs). The purpose of the present study was therefore to identify any resistance-associated changes in miRNA expression in a sensitive and five increasingly drug-resistant Ehrlich ascites tumor (EAT) cell lines, representing different steps in the development of resistance. We used an LNA-enhanced microarray platform to study the global miRNA expression profiles in the six murine EAT cell lines, and identified growth-, hypoxia-, and resistance-specific miRNA patterns. Among the differentially expressed miRNAs, we found the two clusters miR-183∼miR- 96∼miR-182 and miR-200b∼miR-200a∼miR-429 as well as miR-141 to be consistently upregulated in the MDR cell lines, while miR-125b-5p and the two clusters miR-30d∼miR-30b and miR-23b∼miR-27b∼miR-24-1 were downregulated in most of the resistant EAT cells. Several of the target genes for these miRNAs-including Zeb1/Zeb2 and members of the Fox gene family-could contribute to the drug-resistant phenotype, although we did not find that the degree of resistance was directly correlated to any specific changes in miRNA expression. Probably, the observed miRNA expression patterns reflect the underlying genomic instability of the tumor cells, and further studies are needed to explore how the highly complex regulatory miRNA networks contribute to the development of MDR. © 2011 American Chemical Society. Source

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