Repeatable, Inducible Micro-RNA-Based Technology Tightly Controls Liver Transgene Expression

Oprea, Iulian; Viola, Joana R.; Moreno, Pedro M. D.; Simonson, Oscar E.; Rodin, Sergey; Teller, Nathalie; Tryggvason, Karl; Lundin, Karin E.; Girnita, Leonard; Smith, C. I. Edvard

doi:10.1038/mtna.2014.25

Cited by 3 publications

(3 citation statements)

References 46 publications

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“…A fragment of the human PCSK9 genomic sequence including exon 7 to exon 9 was PCR amplified and cloned into the plasmid pLIVE.Luc [35] between the NheI and AgeI restriction sites, generating the reporter vector pLIVE.PCSK9.Luc (see Supplementary data, Fig. C1).…”

Section: In Vivo Experimentsmentioning

confidence: 99%

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form

Rocha

Wiklander

Larsson

et al. 2015

Journal of Molecular and Cellular Cardiology

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Hypercholesterolemia is a medical condition often characterized by high levels of low-density lipoprotein cholesterol (LDL-C) in the blood. Despite the available therapies, not all patients show sufficient responses, especially those with very high levels of LDL-C or those with familial hypercholesterolemia. Regulation of plasma cholesterol levels is very complex and several proteins are involved (both receptors and enzymes). From these, the proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising pharmacologic target. The objective of this work is to develop a new approach to inactivate PCSK9 by splice-switching oligonucleotides (SSOs), converting the normal splice form to a natural, less abundant and inactive, splice variant. For this purpose, a new RNA therapeutic approach for hypercholesterolemia based on SSOs was developed for modulation of the splice pattern of human PCSK9 pre-mRNA. Our results show an increase of the selected splice form at both the mRNA and protein level when compared to non-treated Huh7 and HepG2 cell lines, with concomitant increase of the protein level of the low-density lipoprotein receptor (LDLR) demonstrating the specificity and efficiency of the system. In vivo, full conversion to the splice form was achieved in a reporter system when mice were treated with the specific oligonucleotide, thus further indicating the therapeutic potential of the approach. In conclusion, PCSK9 activity can be modulated by splice-switching through an RNA therapeutic approach. The tuning of the natural active to non-active isoforms represents a physiological way of regulating the cholesterol metabolism, by controlling the amount of LDL receptor available and the rate of LDL-cholesterol clearance.

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Section: In Vivo Experimentsmentioning

confidence: 99%

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form

Rocha

Wiklander

Larsson

et al. 2015

Journal of Molecular and Cellular Cardiology

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“…Recently, the implementation of miRs or antagomiRs have been suggested as novel therapeutic options against various human diseases including cardiovascular diseases [ 38 , 39 ], viral infections [ 40 ] and tumors [ 41 ]. Indeed this strategy is currently tested in animal models or even in clinical trials like Miravirsen (SPC3649/Santaris Pharma A/S) for the treatment of chronic hepatitis C virus infections [ 42 ] or MRX34 (Mirna Therapeutics), which recently entered multicenter open-label phase I clinical trials for patients with liver cancer [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Identification of novel microRNAs regulating HLA-G expression and investigating their clinical relevance in renal cell carcinoma

et al. 2016

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The non-classical human leukocyte antigen G (HLA-G) is expressed at a high frequency in renal cell carcinoma (RCC) and is associated with a higher tumor grade and a poor clinical outcome. This might be caused by the HLA-G-mediated inhibition of the cytotoxicity of T and NK cells. Therefore a selective targeting of HLA-G might represent a powerful strategy to enhance the immunogenicity of RCC lesions. Recent studies identified a number of HLA-G-regulating microRNAs (miRs) and demonstrated an inverse expression of some of these miRs with HLA-G in RCC in vitro and in vivo. However, it was postulated that further miRs might exist contributing to the tightly controlled selective HLA-G expression.By application of a miR enrichment assay (miTRAP) in combination with in silico profiling two novel HLA-G-regulatory miRs, miR-548q and miR-628-5p, were identified. Direct interactions of both miRs with the 3′ untranslated region of HLA-G were confirmed with luciferase reporter gene assays. In addition, qPCR analyses and immunohistochemical staining revealed an inverse, expression of miR-628-5p, but not of miR-548q to the HLA-G protein in primary RCC lesions and cell lines. Stable overexpression of miR-548q and miR-628-5p caused a downregulation of HLA-G mRNA and protein. This leads in case of miR-548q to an enhanced NK cell-mediated HLA-G-dependent cytotoxicity, which could be reverted by ILT2 blockade suggesting a control of the immune effector cell activity at least by this miR. The identification of two novel HLA-G-regulatory miRs extends the number of HLA-G-relevant miRs tuning the HLA-G expression and might serve as future therapeutic targets.

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“…In this study, by adding four complementary sites of microRNA-122 at the 3′UTR of the vector, transgene expression was maintained at very low levels. The expression of the transgene could be restored repeatedly by using a microRNA antagonist, even 6 months after vector administration in vivo [ 48 ].…”

Section: Non-viral Vectorsmentioning

confidence: 99%

Progresses towards safe and efficient gene therapy vectors

et al. 2015

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The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

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Repeatable, Inducible Micro-RNA-Based Technology Tightly Controls Liver Transgene Expression

Cited by 3 publications

References 46 publications

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form

Identification of novel microRNAs regulating HLA-G expression and investigating their clinical relevance in renal cell carcinoma

Progresses towards safe and efficient gene therapy vectors

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