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Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics

Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exp...

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Autores principales: Barbon, Elena, Ferrarese, Mattia, van Wittenberghe, Laetitia, Sanatine, Peggy, Ronzitti, Giuseppe, Collaud, Fanny, Colella, Pasqualina, Pinotti, Mirko, Mingozzi, Federico
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155329/
https://www.ncbi.nlm.nih.gov/pubmed/27898092
http://dx.doi.org/10.1038/mtna.2016.97
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author Barbon, Elena
Ferrarese, Mattia
van Wittenberghe, Laetitia
Sanatine, Peggy
Ronzitti, Giuseppe
Collaud, Fanny
Colella, Pasqualina
Pinotti, Mirko
Mingozzi, Federico
author_facet Barbon, Elena
Ferrarese, Mattia
van Wittenberghe, Laetitia
Sanatine, Peggy
Ronzitti, Giuseppe
Collaud, Fanny
Colella, Pasqualina
Pinotti, Mirko
Mingozzi, Federico
author_sort Barbon, Elena
collection PubMed
description Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X) to integrate human factor IX (hFIX) minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt) or mutated (SChFIXex5-2C). In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV) vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies.
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spelling pubmed-51553292016-12-20 Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics Barbon, Elena Ferrarese, Mattia van Wittenberghe, Laetitia Sanatine, Peggy Ronzitti, Giuseppe Collaud, Fanny Colella, Pasqualina Pinotti, Mirko Mingozzi, Federico Mol Ther Nucleic Acids Original Article Disease-causing splicing mutations can be rescued by variants of the U1 small nuclear RNA (U1snRNAs). However, the evaluation of the efficacy and safety of modified U1snRNAs as therapeutic tools is limited by the availability of cellular and animal models specific for a given mutation. Hence, we exploited the hyperactive Sleeping Beauty transposon system (SB100X) to integrate human factor IX (hFIX) minigenes into genomic DNA in vitro and in vivo. We generated stable HEK293 cell lines and C57BL/6 mice harboring splicing-competent hFIX minigenes either wild type (SChFIX-wt) or mutated (SChFIXex5-2C). In both models the SChFIXex5-2C variant, found in patients affected by Hemophilia B, displayed an aberrant splicing pattern characterized by exon 5 skipping. This allowed us to test, for the first time in a genomic DNA context, the efficacy of the snRNA U1-fix9, delivered with an adeno-associated virus (AAV) vector. With this approach, we showed rescue of the correct splicing pattern of hFIX mRNA, leading to hFIX protein expression. These data validate the SB100X as a versatile tool to quickly generate models of human genetic mutations, to study their effect in a stable DNA context and to assess mutation-targeted therapeutic strategies. Nature Publishing Group 2016-11 2016-11-29 /pmc/articles/PMC5155329/ /pubmed/27898092 http://dx.doi.org/10.1038/mtna.2016.97 Text en Copyright © 2016 Official journal of the American Society of Gene & Cell Therapy http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
spellingShingle Original Article
Barbon, Elena
Ferrarese, Mattia
van Wittenberghe, Laetitia
Sanatine, Peggy
Ronzitti, Giuseppe
Collaud, Fanny
Colella, Pasqualina
Pinotti, Mirko
Mingozzi, Federico
Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics
title Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics
title_full Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics
title_fullStr Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics
title_full_unstemmed Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics
title_short Transposon-mediated Generation of Cellular and Mouse Models of Splicing Mutations to Assess the Efficacy of snRNA-based Therapeutics
title_sort transposon-mediated generation of cellular and mouse models of splicing mutations to assess the efficacy of snrna-based therapeutics
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155329/
https://www.ncbi.nlm.nih.gov/pubmed/27898092
http://dx.doi.org/10.1038/mtna.2016.97
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