Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles
Résumé
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 DeltaF508 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.
Mots clés
Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/metabolism
Genetic/*methods
Epithelial Cells/*metabolism/pathology
Exosomes/chemistry/metabolism
Extracellular Vesicles/*chemistry/metabolism
Green Fluorescent Proteins/genetics/metabolism
Genetic Therapy/*methods
Genetic Vectors/chemistry/metabolism
Humans
Gene Expression
Adenoviruses
Messenger/*genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism
Respiratory Mucosa/metabolism/pathology
RNA
Transduction
Tumor
Cell Line
Cell-Derived Microparticles/*chemistry/metabolism
Human/genetics/metabolism
HEK293 Cells