Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells

2002 ◽  
Vol 5 (1) ◽  
pp. 38-48 ◽  
Author(s):  
Isabelle Fajac ◽  
Guiti Thévenot ◽  
Laurent Bédouet ◽  
Claire Danel ◽  
Marc Riquet ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Transfer Efficiency ◽  
Airway Epithelial ◽  
Polymer Complexes ◽  
Gene Transfer Efficiency

scholarly journals Limited Entry of Adenovirus Vectors into Well-Differentiated Airway Epithelium Is Responsible for Inefficient Gene Transfer

1998 ◽  
Vol 72 (7) ◽  
pp. 6014-6023 ◽  
Author(s):  
Raymond J. Pickles ◽  
Douglas McCarty ◽  
Hirotoshi Matsui ◽  
Pádraig J. Hart ◽  
Scott H. Randell ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Adenovirus Vector ◽  
Airway Epithelial Cells ◽  
Transfer Efficiency ◽  
Apical Plasma Membrane ◽  
Airway Epithelial ◽  
Gene Transfer Efficiency ◽  
Well Differentiated

ABSTRACT Investigations of the efficiency and safety of human adenovirus vector (AdV)-mediated gene transfer in the airways of patients with cystic fibrosis (CF) in vivo have demonstrated little success in correcting the CF bioelectrical functional defect, reflecting the inefficiency of AdV-mediated gene transfer to the epithelial cells that line the airway luminal surface. In this study, we demonstrate that low AdV-mediated gene transfer efficiency to well-differentiated (WD) cultured airway epithelial cells is due to three distinct steps in the apical membrane of the airway epithelial cells: (i) the absence of specific adenovirus fiber-knob protein attachment receptors; (ii) the absence of αvβ3/5 integrins, reported to partially mediate the internalization of AdV into the cell cytoplasm; and (iii) the low rate of apical plasma membrane uptake pathways of WD airway epithelial cells. Attempts to increase gene transfer efficiency by increasing nonspecific attachment of AdV were unsuccessful, reflecting the inability of the attached vector to enter (penetrate) WD cells via nonspecific entry paths. Strategies to improve the efficiency of AdV for the treatment of CF lung disease will require methods to increase the attachment of AdV to and promote its internalization into the WD respiratory epithelium.

Genetic modification of AAV2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells

2008 ◽  
Vol 0 (ja) ◽  
pp. 081015093227032
Author(s):  
April F White ◽  
Marina Mazur ◽  
Eric J Sorscher ◽  
Kurt Zinn ◽  
Selvarangan Ponnazhagan
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Genetic Modification ◽  
Airway Epithelial Cells ◽  
Transfer Efficiency ◽  
Airway Epithelial ◽  
Gene Transfer Efficiency ◽  
Human Airway ◽  
Human Airway Epithelial Cells

scholarly journals Immortalization and transformation of primary human airway epithelial cells by gene transfer

Oncogene ◽  
2002 ◽  
Vol 21 (29) ◽  
pp. 4577-4586 ◽  
Author(s):  
Ante S Lundberg ◽  
Scott H Randell ◽  
Sheila A Stewart ◽  
Brian Elenbaas ◽  
Kimberly A Hartwell ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
Human Airway Epithelial Cells

scholarly journals Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

1998 ◽  
Vol 72 (11) ◽  
pp. 8904-8912 ◽  
Author(s):  
S. Teramoto ◽  
J. S. Bartlett ◽  
D. McCarty ◽  
X. Xiao ◽  
R. J. Samulski ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Gene Transfer ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Adeno Associated Virus ◽  
Differentiated Cells ◽  
Factors Influencing

ABSTRACT Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZgene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.

scholarly journals Respiratory Syncytial Virus Infection of Human Airway Epithelial Cells Is Polarized, Specific to Ciliated Cells, and without Obvious Cytopathology

2002 ◽  
Vol 76 (11) ◽  
pp. 5654-5666 ◽  
Author(s):  
Liqun Zhang ◽  
Mark E. Peeples ◽  
Richard C. Boucher ◽  
Peter L. Collins ◽  
Raymond J. Pickles
Keyword(s):  
Gene Transfer ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cells ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Ciliated Cells ◽  
Transfer Vectors ◽  
Syncytial Virus

ABSTRACT Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.

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