title: Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors.
abstract: Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of reporter gene transfer into immortalized normal (MM-39) and CF (CF-KM4) human airway epithelial gland serous cells using various synthetic vectors: glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using alpha-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using alpha-glycosylated polylysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane. The presence of membrane lectins, i. e., cell surface receptors binding oligosaccharides, was also examined on MM-39 and CF-KM4 cells by assessing the binding and uptake of fluorescein-labeled neoglycoproteins and fluorescein-labeled glycoplexes (glycofectins complexed to plasmid DNA). Among all the neoglycoproteins and glycoplexes tested, those bearing alpha-mannosylated derivatives were most efficiently taken up by both normal and CF gland serous cells. However, alpha-mannosylated polylysine was quite inefficient for gene transfer, indicating that the efficiency of gene transfer is determined both by the uptake of the complexes and also by their intracellular trafficking. Moreover, our results show that an efficient in vitro gene transfer was achieved in human airway gland serous cells with the same synthetic vectors described to efficiently transfect human airway surface epithelial cells.
authors.affiliation: INSERM U380, ICGM, Université René Descartes (Paris V), Paris, France.
authors.forename: J C
authors.initials: JC
authors.lastname: Allo
authors.forename: P
authors.initials: P
authors.lastname: Midoux
authors.forename: M
authors.initials: M
authors.lastname: Merten
authors.forename: E
authors.initials: E
authors.lastname: Souil
authors.forename: J
authors.initials: J
authors.lastname: Lipecka
authors.forename: C
authors.initials: C
authors.lastname: Figarella
authors.forename: M
authors.initials: M
authors.lastname: Monsigny
authors.forename: P
authors.initials: P
authors.lastname: Briand
authors.forename: I
authors.initials: I
authors.lastname: Fajac
mesh_terms: D002461:Cell Line, Transformed; D003550:Cystic Fibrosis / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D014132:Trachea / Q000378:metabolism* / Q000473:pathology
chemical_list: C505032:CFTR protein, human; D037102:Lectins; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator