Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges

Hollensen, Anne Kruse; Thomsen, Rie; Bak, Rasmus O.; Petersen, Charlotte Christie; Ermegaard, Eva Reumert; Aagaard, Lars; Damgaard, Christian Kroun; Mikkelsen, Jacob Giehm

doi:10.1261/rna.061192.117

Cited by 11 publications

(21 citation statements)

References 49 publications

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“…Third-generation lentiviral vectors were produced in HEK293T cells as previously described 34 . One day before transfection, cells were seeded in 10cm dishes at a density of 4 × 10 6 cells/dish.…”

Section: Lentiviral Productionmentioning

confidence: 99%

“…All lentiviral preparations were made in at least triplicates and pooled before determination of viral titers. To determine viral titers of lentiviral preparations, flow cytometric measurements of EGFP expression were used as previously described 34 . One day prior to transduction, L-AN-5 cells were seeded at a density of 5 x 10 5 cells/well in 12-well plates.…”

Section: Lentiviral Productionmentioning

confidence: 99%

“…Subcellular fractionation of nuclear and cytoplasmic RNA was carried out as previously described in 36 . Briefly, cells were washed in PBS, added 800 µl PBS and scraped off.…”

Section: Subcellular Fractionation Of Nuclear and Cytoplasmic Rnamentioning

confidence: 99%

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circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation

Hollensen

Thomsen

Marta

et al. 2019

Preprint

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Circular RNAs (circRNAs) comprise a large class of conserved non-codingRNAs that regulate a number of biological processes, including transcription, alternative splicing, translation and mRNA decay. circRNAs are enriched in the brain and mammalian cells subjected to neuronal differentiation markedly change their circRNA transcriptomes. Here, we have mapped high-confidence circRNA inventories of mouse embryonic stem cells, neuronal progenitor cells and in differentiated glutamatergic neurons and identify hundreds of differentially expressed circRNAs. Among several candidate circRNAs, knockdown of circZNF827 significantly induces expression of key neuronal markers, suggesting that this molecule negatively regulates neuronal differentiation. Using Nanostring analyses we demonstrate that among 770 tested genes linked to known neuronal pathways and neuropathological states, knockdown of circZNF827 deregulates expression of several genes including a robust upregulation of neuronal growth factor receptor (NGFR).We show that NGFR becomes transcriptionally upregulated and that this functionally enhances NGF signalling. Our results suggest that circZNF827, although being highly enriched in the cell cytoplasm, can elicit transcriptional changes, which in turn balances proliferation and neuronal differentiation signalling.

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Section: Lentiviral Productionmentioning

confidence: 99%

Section: Lentiviral Productionmentioning

confidence: 99%

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circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation

Hollensen

Thomsen

Marta

et al. 2019

Preprint

Self Cite

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“…To improve the miRNA suppression potential of the TuD hairpin, Hollensen et al (2017) inserted an RNA element, (Woodchuck hepatitis virus [WHV] post-transcriptional regulatory element [PRE], WPRE) upstream of the TuD hairpin. The WPRE RNA element has been shown to enhance gene expression in vitro and in vivo .…”

Section: Future Developments For Mirna-targeting Therapeutics – New Tmentioning

confidence: 99%

Generation of MicroRNA-34 Sponges and Tough Decoys for the Heart: Developments and Challenges

Bernardo¹,

Gregorevic²,

Ritchie³

et al. 2018

Front. Pharmacol.

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Heart failure (HF) is a debilitating and deadly chronic disease, with almost 50% of patients with HF dying within 5 years of diagnosis. With limited effective therapies to treat or cure HF, new therapies are greatly needed. microRNAs (miRNAs) are small non-coding RNA molecules that are powerful regulators of gene expression and play a key role in almost every biological process. Disruptions in miRNA gene expression has been functionally linked to numerous diseases, including cardiovascular disease. Molecular tools for manipulating miRNA activity have been developed, and there is evidence from preclinical studies demonstrating the potential of miRNAs to be therapeutic targets for cardiovascular disease. For clinical application, miRNA sponges and tough decoys have been developed for more stable suppression and targeted delivery of the miRNA of choice. The aim of this study was to generate miRNA sponges and tough decoys to target miR-34 in the mouse heart. We present data to show that using both approaches we were unable to get significant knockdown of miR-34 or regulate miR-34 target genes in the heart in vivo. We also review recent applications of this method in the heart and discuss further considerations for optimisation in construct design and testing, and the obstacles to be overcome before they enter the clinic.

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“…TuDs are exported out of the nucleus to the cytoplasm via Exportin 5, where they specifically and potently interfere with miRNA activity [23]. Studies have also shown that RNA polymerase II promoters can be used to efficiently express TuD inhibitors within the cell [24,25]. It has been shown that adeno-associated virus-delivered TuDs induced long-term miRNA inhibition in mice [27,28].…”

Section: Introductionmentioning

confidence: 99%

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

et al. 2018

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MicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression through silencing of complementary target mRNAs. miRNAs are involved in many biological processes, including cell proliferation, differentiation, cell signaling and cellular defense responses to infection. Strategies that allow for strong and stable suppression of specific microRNA activity are needed to study miRNA functions and to develop therapeutic intervention strategies aimed at interfering with miRNA activity in vivo. One of these classes of miRNA inhibitors are Tough Decoys (TuD) RNAs, which comprise of an imperfect RNA hairpin structure that harbors two opposing miRNA binding sites. Upon developing TuDs targeting Epstein-Barr virus miRNAs, we observed a strong variation in inhibitory potential between different TuD RNAs targeting the same miRNA. We show that the composition of the ‘bulge’ sequence in the miRNA binding sites has a strong impact on the inhibitory potency of the TuD. Our data implies that miRNA inhibition correlates with the thermodynamic properties of the TuD and that design aimed at lowering the TuD opening energy increases TuD potency. Our study provides specific guidelines for the design and construction of potent decoy-based miRNA inhibitors, which may be used for future therapeutic intervention strategies.

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Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges

Cited by 11 publications

References 49 publications

circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation

circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation

Generation of MicroRNA-34 Sponges and Tough Decoys for the Heart: Developments and Challenges

RNA accessibility impacts potency of Tough Decoy microRNA inhibitors

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