title: Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles.
abstract: Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA(GFP-CFTR)) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR). Both microvesicles and exosomes had the capacity to package and deliver the GFP-CFTR glycoprotein and mRNA(GFP-CFTR) to target cells in a dose-dependent manner. Homologous versus heterologous EV-to-cell transfer was studied, and it appeared that the cellular uptake of EVs was significantly more efficient in homologous transfer. The incubation of CF15 cells, a nasal epithelial cell line homozygous for the ΔF508 CFTR mutation, with microvesicles or exosomes loaded with GFP-CFTR resulted in the correction of the CFTR function in CF cells in a dose-dependent manner. A time-course analysis of EV-transduced CF cells suggested that CFTR transferred as mature glycoprotein was responsible for the CFTR-associated channel activity detected at early times posttransduction, whereas GFP-CFTR translated from exogenous mRNA(GFP-CFTR) was responsible for the CFTR function at later times. Collectively, this study showed the potential application of microvesicles and exosomes as vectors for CFTR transfer and functional correction of the genetic defect in human CF cells.
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .|2 Institut de Biologie et Chimie des Protéines, Unité BMSSI, UMR 5086 CNRS-Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Cyrielle
authors.initials: C
authors.lastname: Vituret
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Kathy
authors.initials: K
authors.lastname: Gallay
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Confort
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Najate
authors.initials: N
authors.lastname: Ftaich
authors.affiliation: 3 Centre Technologique des Microstructures, Université Claude Bernard-Lyon , Villeurbanne, France .
authors.forename: Constantin I
authors.initials: CI
authors.lastname: Matei
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Fabienne
authors.initials: F
authors.lastname: Archer
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Corinne
authors.initials: C
authors.lastname: Ronfort
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Mornex
authors.affiliation: 4 Département de Physiologie Cellulaire & Métabolisme, Centre Médical Universitaire , Geneva, Switzerland .
authors.forename: Marc
authors.initials: M
authors.lastname: Chanson
authors.affiliation: 2 Institut de Biologie et Chimie des Protéines, Unité BMSSI, UMR 5086 CNRS-Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Attilio
authors.initials: A
authors.lastname: Di Pietro
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .
authors.forename: Pierre
authors.initials: P
authors.lastname: Boulanger
authors.affiliation: 1 Viral Infections & Comparative Pathology, UMR-754 UCBL-INRA-EPHE, Université Lyon 1 , Lyon Cedex 07, France .|5 Institut National de la Santé et de la Recherche Médicale , Paris, France .
authors.forename: Saw See
authors.initials: SS
authors.lastname: Hong
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D055252:Cell-Derived Microparticles / Q000737:chemistry* / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D055354:Exosomes / Q000737:chemistry / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism / Q000473:pathology; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C505032:CFTR protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins