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Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting...

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Autores principales: Stenler, Sofia, Wiklander, Oscar PB, Badal-Tejedor, Maria, Turunen, Janne, Nordin, Joel Z, Hallengärd, David, Wahren, Britta, Andaloussi, Samir EL, Rutland, Mark W, Smith, CI Edvard, Lundin, Karin E, Blomberg, Pontus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910003/
https://www.ncbi.nlm.nih.gov/pubmed/24399204
http://dx.doi.org/10.1038/mtna.2013.67
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author Stenler, Sofia
Wiklander, Oscar PB
Badal-Tejedor, Maria
Turunen, Janne
Nordin, Joel Z
Hallengärd, David
Wahren, Britta
Andaloussi, Samir EL
Rutland, Mark W
Smith, CI Edvard
Lundin, Karin E
Blomberg, Pontus
author_facet Stenler, Sofia
Wiklander, Oscar PB
Badal-Tejedor, Maria
Turunen, Janne
Nordin, Joel Z
Hallengärd, David
Wahren, Britta
Andaloussi, Samir EL
Rutland, Mark W
Smith, CI Edvard
Lundin, Karin E
Blomberg, Pontus
author_sort Stenler, Sofia
collection PubMed
description The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.
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spelling pubmed-39100032014-02-03 Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression Stenler, Sofia Wiklander, Oscar PB Badal-Tejedor, Maria Turunen, Janne Nordin, Joel Z Hallengärd, David Wahren, Britta Andaloussi, Samir EL Rutland, Mark W Smith, CI Edvard Lundin, Karin E Blomberg, Pontus Mol Ther Nucleic Acids Original Article The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo. Nature Publishing Group 2014-01 2014-01-07 /pmc/articles/PMC3910003/ /pubmed/24399204 http://dx.doi.org/10.1038/mtna.2013.67 Text en Copyright © 2014 The American Society of Gene & Cell Therapy
spellingShingle Original Article
Stenler, Sofia
Wiklander, Oscar PB
Badal-Tejedor, Maria
Turunen, Janne
Nordin, Joel Z
Hallengärd, David
Wahren, Britta
Andaloussi, Samir EL
Rutland, Mark W
Smith, CI Edvard
Lundin, Karin E
Blomberg, Pontus
Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression
title Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression
title_full Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression
title_fullStr Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression
title_full_unstemmed Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression
title_short Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression
title_sort micro-minicircle gene therapy: implications of size on fermentation, complexation, shearing resistance, and expression
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910003/
https://www.ncbi.nlm.nih.gov/pubmed/24399204
http://dx.doi.org/10.1038/mtna.2013.67
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