Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 1998 Nov;72(11):8904-12.
doi: 10.1128/JVI.72.11.8904-8912.1998.

Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors

S Teramoto  1 , J S Bartlett, D McCarty, X Xiao, R J Samulski, R C Boucher
Affiliations
Comparative Study

Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors

S Teramoto et al. J Virol. 1998 Nov.

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 lacZ gene 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.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Efficiency of AAVβgal-mediated gene transfer to human airway epithelial cells. Values are presented as means ± standard errors of the means (n = 5). Cells were infected for 1 h at 37°C, and the transduction efficiencies were determined by histochemical staining for β-galactosidase activity 2 days after infection, as described in Materials and Methods.
FIG. 2
FIG. 2
The transduction efficiencies of AAV and Ad vectors in primary cultures of human airway epithelial cells (A), CF/T43 cells (B), and CFT1 cells (C). Values are presented as means ± standard errors of the means (n = 3). Experimental conditions were the same as described in the legend to Fig. 1 and are detailed in Materials and Methods.
FIG. 3
FIG. 3
Effect of the presence of Ad on transduction of AAVβgal. (A) Airway epithelial cells infected with AAV or Ad vector, alone or with Ad dl309. Values are presented as means ± standard errors of the means (n = 3). Airway cells were infected with AAVβgal vector (100 TU/cell; 104 particles/cell) or AdCMVβgal vector (100 TU/cell; 2 × 103 particles/cell) in the presence or absence of Ad dl309 (100 IU/cell) for 1 h. Two days later, the transduction efficiencies were determined by staining for β-galactosidase activity as described in Materials and Methods. (B) The relative effect of Ad on AAVβgal transduction of HEK 293 cells, HeLa cells, and human airway epithelial cells. Levels of transduction in the presence of Ad are shown relative to the level of transduction in the absence of Ad, which has been assigned a value of 1. Airway cells were infected with the AAVβgal vector (100 TU/cell; 104 particles/cell) or the AdCMVβgal vector (100 TU/cell; 2 × 103 particles/cell) in the presence or absence of Ad dl309 (100 IU/cell) as described for panel A. To allow for increased transduction in the presence of Ad, HEK 293 and HeLa cells were infected with 0.1 particle of AAVβgal vector or 0.5 particle of AdCMVβgal per cell in the presence or absence of Ad dl309 (50 particles/cell).
FIG. 4
FIG. 4
Relationship between Ad dose and enhancement of AAVβgal transduction in primary cultures of human airway epithelial cells (A), CF/T43 cells (B), and CFT1 cells (C). Cells were infected for 1 h at 37°C, and the transduction efficiencies were determined by histochemical staining for β-galactosidase activity 24 h later. Each value is presented as the mean ± the standard error of the mean (n = 3).
FIG. 5
FIG. 5
Transduction of airway epithelial cells with rAAV in the presence of different Ad mutants. Values are presented as means ± standard errors of the means (n = 3). Vector AAVβgal (104 particles/cell) was administered to cells for 1 h in the presence or absence of Ad dl309, UV- and psoralen-inactivated Ad dl309, E4− Ad, or E4− Ad ORF6+ 6/7+ (each at 1,000 particles/cells). At 24 h after infection, the cells were stained for β-galactosidase activity as described in Materials and Methods.
FIG. 6
FIG. 6
Effect of prolonged vector incubation time on the transduction of primary cultures of human airway epithelial cells (A), CF/T43 cells (B), and CFT1 cells (C) by AAVβgal. Values are presented as means ± standard errors of the means (n = 3). Cells were incubated with the vector for the indicated period of time, washed, cultured for an additional 48 h in the absence of vector, and stained for β-galactosidase activity to determine the efficiency of transduction.
FIG. 7
FIG. 7
Activation of rAAV vector over time in culture. Primary human airway epithelial cells from CF patients (A), CF/T43 cells (B), and CFT1 cells (C) were infected with AAVβgal for 1 h at 37°C. Transduction efficiencies were determined on the indicated days postinfection by staining for β-galactosidase activity as described in Materials and Methods. Values are presented as means ± standard errors of the means (n = 3).
FIG. 8
FIG. 8
Effect of Ad on AAVβgal transduction of primary airway cells from CF patients (A), CF/T43 cells (B), or CFT1 cells (C) at various times postinfection. Cells were initially infected with AAVβgal for 1 h at 37°C, washed, and then exposed to Ad dl309, UV- and psoralen-inactivated Ad dl309, E4− Ad, or E4− ORF6+ 6/7+ Ad for 1 h at the indicated times post-rAAV infection. All cells were stained for LacZ activity 48 h following rAAV infection as described in the text. Values represent the means of data from triplicate experiments.
FIG. 9
FIG. 9
Effect of Ad and DNA-damaging agents on rAAV transduction of airway epithelial cell lines. (A) The indicated cell lines were infected with AAVβgal (1,000 particles/cell) for 1 h at 37°C and then treated with the indicated agents and stained for β-galactosidase activity following 48 h in culture, as described in Materials and Methods. (B) The cells were cultured for 48 h after infection, prior to treatment with the indicated agents, and then cultured for an additional 48 h prior to assessment of transduction.
FIG. 10
FIG. 10
Histological sections of freshly excised tissue from CF patients. Human tracheal specimens were cut into 0.25-cm2 samples and infected with AAVβgal for 30 min. At 48 h after exposure, specimens were fixed with 0.5% glutaraldehyde and stained for β-galactosidase activity as described in Materials and Methods. The sections were counterstained with hematoxylin and eosin. Regions of the epithelium containing well-differentiated columnar cells were not transduced by AAVβgal (A and B), whereas areas of the epithelium that were injured and had exposed cuboidal, basal-like cells were moderately well transduced by this vector (C and D). Magnifications: A, C, and D, ×250; B, ×1,000.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexander I E, Russell D W, Miller A D. DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors. J Virol. 1994;68:8282–8287. - PMC - PubMed
    1. Bartlett J S, Quattrocchi K B, Samulski R J. The development of adeno-associated virus as a vector for cancer gene therapy. In: Sobol R E, Scanlon K J, editors. The Internet book of gene therapy: cancer therapeutics. Stamford, Conn: Appleton & Lange; 1995. pp. 27–40.
    1. Brody S L, Crystal R G. Adenovirus-mediated in vivo gene transfer. Ann N Y Acad Sci. 1994;31:90–101. - PubMed
    1. Conrad C K, Allen S S, Afione S A, Reynolds T C, Beck S E, Fee-Maki M, Barrazza-Ortiz X, Adams R, Askin F B, Carter B J, Guggino W B, Flotte T R. Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther. 1996;3:658–668. - PubMed
    1. Cotten M, Wagner E, Zatloukal K, Phillips S, Curiel D T, Birnstiel M L. High-efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles. Proc Natl Acad Sci USA. 1992;89:6094–6098. - PMC - PubMed

Publication types

Substances

-