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Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition

BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be eluci...

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Autores principales: Bire, Solenne, Gosset, David, Jégot, Gwenhael, Midoux, Patrick, Pichon, Chantal, Rouleux-Bonnin, Florence
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849706/
https://www.ncbi.nlm.nih.gov/pubmed/24070093
http://dx.doi.org/10.1186/1472-6750-13-75
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author Bire, Solenne
Gosset, David
Jégot, Gwenhael
Midoux, Patrick
Pichon, Chantal
Rouleux-Bonnin, Florence
author_facet Bire, Solenne
Gosset, David
Jégot, Gwenhael
Midoux, Patrick
Pichon, Chantal
Rouleux-Bonnin, Florence
author_sort Bire, Solenne
collection PubMed
description BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.
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spelling pubmed-38497062013-12-05 Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition Bire, Solenne Gosset, David Jégot, Gwenhael Midoux, Patrick Pichon, Chantal Rouleux-Bonnin, Florence BMC Biotechnol Research Article BACKGROUND: Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. RESULTS: The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. CONCLUSION: Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking. BioMed Central 2013-09-26 /pmc/articles/PMC3849706/ /pubmed/24070093 http://dx.doi.org/10.1186/1472-6750-13-75 Text en Copyright © 2013 Bire et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Bire, Solenne
Gosset, David
Jégot, Gwenhael
Midoux, Patrick
Pichon, Chantal
Rouleux-Bonnin, Florence
Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition
title Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition
title_full Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition
title_fullStr Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition
title_full_unstemmed Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition
title_short Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition
title_sort exogenous mrna delivery and bioavailability in gene transfer mediated by piggybac transposition
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849706/
https://www.ncbi.nlm.nih.gov/pubmed/24070093
http://dx.doi.org/10.1186/1472-6750-13-75
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