Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry

Abstract

The limitations of adeno-associated virus (AAV)-mediated vectors for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with glycosidases. These results indicate that transduction of airway epithelia by AAV vectors is limited by entry and reinforce the importance of a physical barrier on the airway surface.

FIG. 1

Levels of transgene expression after infection of human airway cultures in transwell cultures. (A) The status of differentiation is an important determinant of transduction efficiency. Cells were infected from the apical side at different time points after seeding onto the transwells, and numbers of transgene-expressing cells were determined 7 days after infection. (B) In polarized epithelium, transduction efficiency is dependent on the site of vector application. AAV was applied to fully differentiated epithelium (after 3 weeks of culture) from the apical or basolateral side, and the expression of the transgene was assayed over 40 days. Recombinant adenovirus (rAd) coding for GFP (Ad5.010–CMV–eGFP) was applied to the apical or basolateral side of the cultures (10⁶ particles/200 μl). This treatment did not result in a change in the R_t (data not shown). (C) Treatment with glycerol (10%) or pretreatment with 0.05 g of trypsin/liter–0.05 mM EDTA for 2 min, followed by three washes, increased the transduction efficiency of AAV vectors. Coinfection with wild-type adenovirus (serotype 5) (Ad) resulted in increased numbers of positive cells compared to no treatment (normal). (D) To determine whether the apical application of glycerol or trypsin to differentiated cultures resulted in temporal disruption of the apical tight junctions, R_t was measured up to 24 h after the application of the agent. Trypsinization did not decrease the R_t compared to the medium control (normal); however, glycerol resulted in a significant decrease in R_t.

FIG. 2

Southern blots of total cellular DNA extracted after 40 days of culture (except for lane 1, where cells were harvested 14 days after seeding and infection) were hybridized to radiolabeled eGFP cDNA as a probe. Shown are amounts of viral DNA in cells after infection immediately after seeding of the cells into the transwells (lane 1); after infection of differentiated cells by apical application with no further treatment (lane 2), apical application together with 10% glycerol (lane 3), basolateral application (lane 4), or apical application after predigestion with trypsin for 2 min (lane 5); and after infection of differentiated cells that had been pretreated with neuraminidase (lane 6) or endoglycosidase H (lane 7) compared to no treatment (lane 8). DS, double-stranded viral DNA; SS, single-stranded viral DNA.

FIG. 3

Micrographs showing representative areas of the epithelial layer using UV illumination. Cells were infected with a standard dose of 10¹¹ genome particles of AAV vector coding for eGFP. All cultures were fully differentiated at the time of infection. Different treatments are indicated at the top; the times after infection are indicated on the right. Bar, 100 μm.

FIG. 4

Negatively charged carbohydrates inhibit the transduction efficiency of AAV vector added to the apical side of differentiated cultures. The addition of mucin (pig stomach mucin; final concentration, 1 mg/ml) or heparin (5 μg/ml) (both from Sigma) resulted in inhibition of transduction, whereas pretreatment with several glycosidases for 1 h enhanced transduction. N-glycosidase F (final concentration, 5 U/ml), neuraminidase (0.01 mg/ml), N-glycosidase A (1 mU/ml), or endoglycosidase H (0.2 U/ml) (all from Boehringer Mannheim) was diluted in medium and applied to the apical surface of the epithelium for 1 h. After three washes, the medium containing the virus was added. ∗∗, statistically significant difference between the specified group and the “no treatment” group (P < 0.005).