Cargando…

Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene

Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Ad...

Descripción completa

Detalles Bibliográficos
Autores principales: De Rocco, Davide, Pompili, Barbara, Castellani, Stefano, Morini, Elena, Cavinato, Luca, Cimino, Giuseppe, Mariggiò, Maria A, Guarnieri, Simone, Conese, Massimo, Del Porto, Paola, Ascenzioni, Fiorentina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979583/
https://www.ncbi.nlm.nih.gov/pubmed/29673202
http://dx.doi.org/10.3390/ijms19041220
_version_ 1783327731336871936
author De Rocco, Davide
Pompili, Barbara
Castellani, Stefano
Morini, Elena
Cavinato, Luca
Cimino, Giuseppe
Mariggiò, Maria A
Guarnieri, Simone
Conese, Massimo
Del Porto, Paola
Ascenzioni, Fiorentina
author_facet De Rocco, Davide
Pompili, Barbara
Castellani, Stefano
Morini, Elena
Cavinato, Luca
Cimino, Giuseppe
Mariggiò, Maria A
Guarnieri, Simone
Conese, Massimo
Del Porto, Paola
Ascenzioni, Fiorentina
author_sort De Rocco, Davide
collection PubMed
description Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease.
format Online
Article
Text
id pubmed-5979583
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-59795832018-06-10 Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene De Rocco, Davide Pompili, Barbara Castellani, Stefano Morini, Elena Cavinato, Luca Cimino, Giuseppe Mariggiò, Maria A Guarnieri, Simone Conese, Massimo Del Porto, Paola Ascenzioni, Fiorentina Int J Mol Sci Article Improving the efficacy of gene therapy vectors is still an important goal toward the development of safe and efficient gene therapy treatments. S/MAR (scaffold/matrix attached region)-based vectors are maintained extra-chromosomally in numerous cell types, which is similar to viral-based vectors. Additionally, when established as an episome, they show a very high mitotic stability. In the present study we tested the idea that addition of an S/MAR element to a CFTR (cystic fibrosis transmembrane conductance regulator) expression vector, may allow the establishment of a CFTR episome in bronchial epithelial cells. Starting from the observation that the S/MAR vector pEPI-EGFP (enhanced green fluorescence protein) is maintained as an episome in human bronchial epithelial cells, we assembled the CFTR vector pBQ-S/MAR. This vector, transfected in bronchial epithelial cells with mutated CFTR, supported long term wt CFTR expression and activity, which in turn positively impacted on the assembly of tight junctions in polarized epithelial cells. Additionally, the recovery of intact pBQ-S/MAR, but not the parental vector lacking the S/MAR element, from transfected cells after extensive proliferation, strongly suggested that pBQ-S/MAR was established as an episome. These results add a new element, the S/MAR, that can be considered to improve the persistence and safety of gene therapy vectors for cystic fibrosis pulmonary disease. MDPI 2018-04-17 /pmc/articles/PMC5979583/ /pubmed/29673202 http://dx.doi.org/10.3390/ijms19041220 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
De Rocco, Davide
Pompili, Barbara
Castellani, Stefano
Morini, Elena
Cavinato, Luca
Cimino, Giuseppe
Mariggiò, Maria A
Guarnieri, Simone
Conese, Massimo
Del Porto, Paola
Ascenzioni, Fiorentina
Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene
title Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene
title_full Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene
title_fullStr Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene
title_full_unstemmed Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene
title_short Assembly and Functional Analysis of an S/MAR Based Episome with the Cystic Fibrosis Transmembrane Conductance Regulator Gene
title_sort assembly and functional analysis of an s/mar based episome with the cystic fibrosis transmembrane conductance regulator gene
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979583/
https://www.ncbi.nlm.nih.gov/pubmed/29673202
http://dx.doi.org/10.3390/ijms19041220
work_keys_str_mv AT deroccodavide assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT pompilibarbara assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT castellanistefano assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT morinielena assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT cavinatoluca assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT ciminogiuseppe assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT mariggiomariaa assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT guarnierisimone assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT conesemassimo assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT delportopaola assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene
AT ascenzionifiorentina assemblyandfunctionalanalysisofansmarbasedepisomewiththecysticfibrosistransmembraneconductanceregulatorgene