Magnetically guided lentiviral-mediated transduction of airway epithelial cells.
BACKGROUND
Lentiviral (LV) vectors are able to only slowly and inefficiently transduce nondividing cells such as those of the airway epithelium. To address this issue, we have exploited the magnetofection technique in in vitro models of airway epithelium.


METHODS
Magnetofectins were formed by noncovalent interaction between LV particles and polycation-coated iron oxide nanoparticles. Efficiency of LV-mediated transduction (as evaluated through green fluorescent protein (GFP) expression by cytofluorimetric analysis) was measured in bronchial epithelial cells in the presence or absence of a magnetic field. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release; cell monolayer integrity by measurement of transepithelial resistance (TER) and evaluation of correct zonula occludens-1 (ZO-1) localization at tight junctions (TJs) by immunofluorescence and confocal microscopy.


RESULTS
In nonpolarized cells, magnetofectins enhanced LV-mediated transduction at multiplicity of infection (MOI) of 50 up to 3.9-fold upon a 24-h incubation, to levels that approached those achieved at MOI of 200 for LV alone, in the presence or absence of the magnetic field. Magnetofection significantly increased the percentage of transduced cells up to 186-fold already after 15 min of incubation. In polarized cells, magnetofection increased GFP+ cells up to 24-fold compared to LV alone. Magnetofection did not enhance LDH release and slightly altered TER but not ZO-1 localization at the TJs.


CONCLUSIONS
We conclude that magnetofection can facilitate in vitro LV-mediated transduction of airway epithelial cells, in the absence of overt cytotoxicity and maintaining epithelial integrity, by lowering the necessary vector dose and reducing the incubation time required to achieve efficient transduction.
Orlando
Clara
C
Institute for Experimental Treatment of Cystic Fibrosis, HS Raffaele, Milan, Italy.
Castellani
Stefano
S
Mykhaylyk
Olga
O
Copreni
Elena
E
Zelphati
Olivier
O
Plank
Christian
C
Conese
Massimo
M
D016764:Cell Polarity; D002470:Cell Survival; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008280:Magnetics; D008565:Membrane Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase