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. 2010 Aug;84(15):7770-81.
doi: 10.1128/JVI.00346-10. Epub 2010 May 26.

Respiratory syncytial virus engineered to express the cystic fibrosis transmembrane conductance regulator corrects the bioelectric phenotype of human cystic fibrosis airway epithelium in vitro

Anna R Kwilas  1 , Mark A Yednak, Liqun Zhang, Rachael Liesman, Peter L Collins, Raymond J Pickles, Mark E Peeples
Affiliations

Respiratory syncytial virus engineered to express the cystic fibrosis transmembrane conductance regulator corrects the bioelectric phenotype of human cystic fibrosis airway epithelium in vitro

Anna R Kwilas et al. J Virol. 2010 Aug.

Abstract

Cystic fibrosis (CF) is the most common lethal recessive genetic disease in the Caucasian population. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that is normally expressed in ciliated airway epithelial cells and the submucosal glands of the lung. Since the CFTR gene was first characterized in 1989, a major goal has been to develop an effective gene therapy for CF lung disease, which has the potential to ameliorate morbidity and mortality. Respiratory syncytial virus (RSV) naturally infects the ciliated cells in the human airway epithelium. In addition, the immune response mounted against an RSV infection does not prevent subsequent infections, suggesting that an RSV-based vector might be effectively readministered. To test whether the large 4.5-kb CFTR gene could be expressed by a recombinant RSV and whether infectious virus could be used to deliver CFTR to ciliated airway epithelium derived from CF patients, we inserted the CFTR gene into four sites in a recombinant green fluorescent protein-expressing RSV (rgRSV) genome to generate virus expressing four different levels of CFTR protein. Two of these four rgRSV-CFTR vectors were capable of expressing CFTR with little effect on viral replication. rgRSV-CFTR infection of primary human airway epithelial cultures derived from CF patients resulted in expression of CFTR protein that was properly localized at the luminal surface and corrected the chloride ion channel defect in these cells.

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Figures

FIG. 1.
FIG. 1.
rgRSV-CFTR constructs. The CFTR gene, flanked by the RSV GS and GE, was inserted into the rgRSV cDNA at 4 positions. (A) Sequences flanking the CFTR gene unit in the four constructs. (B) Schematic of CFTR locations within rgRSV-CFTR genomes. The constructs are named for the position of the CFTR gene relative to the viral promoter at the 3′ end of the genome.
FIG. 2.
FIG. 2.
CFTR expression from rgRSV-CFTR. A Western blot was performed to detect CFTR protein in HeLa cells infected with rgRSV-CFTR vectors. Band C (∼190 kDa) represents full-length, mature CFTR. Band B (∼150 kDa) represents immature CFTR. Endogenous CFTR from Calu3 cells was used as a positive control (lane 5). Lysates from mock-infected HeLa cells and rgRSV-inoculated HeLa cells were negative controls (lanes 6 and 7). Shown is a representative blot from four experiments.
FIG. 3.
FIG. 3.
Growth analysis of rgRSV-CFTR vectors. Shown are growth curves for rgRSV-CFTR10 and rgRSV-CFTR12 compared to parental rgRSV. Duplicate wells of HeLa cells were inoculated at an MOI of 0.1 with either rgRSV, rgRSV-CFTR10, or rgRSV-CFTR12. Virus was harvested at the indicated times and titrated.
FIG. 4.
FIG. 4.
rgRSV-CFTR infection of CF HAE. Quadruplicate CF HAE cultures were inoculated with 2.0 × 105 PFU, 2.0 × 105 PFU, and 1.0 × 106 PFU of rgRSV, rgRSV-CFTR10, or rgRSV-CFTR12, respectively. (A) At 24 h postinoculation, GFP expression was detectable in cultures infected with rgRSV, rgRSV-CFTR10, and rgRSV-CFTR12, indicating infection (representative images are shown). (B) The percentages of GFP-positive cells at 24 h p.i. were quantified, and the results are displayed as means and standard deviations. The results depicted are representative of two experiments, each including quadruplicate cultures for each experimental condition.
FIG. 5.
FIG. 5.
Expression of CFTR mRNA in CF HAE cultures infected with rgRSV, rgRSV-CFTR10, or rgRSV-CFTR12 (2 × 105 PFU, 2 × 105 PFU, and 1 × 106 PFU, respectively) or mock infected. Total mRNA was collected from infected and mock-infected cultures 48 h postinoculation (n = 4). (A) The amount of CFTR mRNA present in infected cultures was compared to that in mock-treated CF HAE cultures. (B) The amount of viral N protein mRNA present in cultures infected with the rgRSV-CFTR vectors was compared to that in rgRSV-infected cultures. All results are shown as means and standard deviations. The asterisks indicate a statistically significant difference (P value < 0.05) as determined by an unpaired Student t test. The results depicted are representative of two experiments, each including quadruplicate cultures for each experimental condition.
FIG. 6.
FIG. 6.
CFTR expression and localization in rgRSV-CFTR-infected CF HAE cultures. At 48 h postinoculation, CF HAE cultures that were mock infected or infected with rgRSV, rgRSV-CFTR10, or rgRSV-CFTR12, as indicated, were sectioned and stained for CFTR with MAb 596, followed by a goat anti-mouse Ig-Alexafluor 568-conjugated secondary antibody (red). Nuclei were stained with Hoechst 33342 (blue). Representative images are shown.
FIG. 7.
FIG. 7.
Ion transport properties of CF HAE cultures following inoculation with rgRSV-CFTRs. CF HAE cultures were inoculated with rgRSV, rgRSV-CFTR10, or rgRSV-CFTR12 (2 × 105 PFU, 2 × 105 PFU, and 1 × 106 PFU, respectively) or were mock infected and were measured for ion transport properties in Ussing chambers at 48 h p.i. (A) Representative traces of the ISC responses (y axis) in mock-, rgRSV-, and rgRSV-CFTR-infected cultures in response to sequential treatment with amiloride (Amil) to inhibit ENaCs, forskolin (Fskl) to activate CFTR activity, CFTR172 to inhibit CFTR activity, and UTP to stimulate Ca2+-activated Cl− channels as a positive control. (B to D) Maximum changes in ISC (ΔISC) in response to individual treatments. (B) ΔISC of CF HAE after amiloride treatment. (C) Forskolin-induced ΔISC of CF HAE, indicating the presence of functional CFTR. (D) ΔISC of CF HAE following treatment with CFTR172. These results showed that the magnitude of forskolin-activated changes in CF HAE infected with the RSV-CFTRs was comparable to that in non-CF HAE (n = 7). All results are shown as means and standard deviations. “ns” indicates a difference that is not statistically significant (P value > 0.05). The asterisk indicates a difference that is statistically significant (P value < 0.05), as determined by an unpaired Student t test. The results depicted are representative of two experiments, each including quadruplicate cultures for each experimental condition.
FIG. 8.
FIG. 8.
ASL measurements of CF HAE 48 h after infection with rgRSV, rgRSV-CFTR10, or rgRSV-CFTR12 (2 × 105 PFU, 2 × 105 PFU, and 1 × 106 PFU, respectively) or mock infection. Each bar represents three measurements on each of four cultures. All results are shown as means and standard deviations. “ns” indicates a difference that is not statistically significant (P value > 0.05). The asterisk indicates a statistically significant difference (P value < 0.05), as determined by an unpaired Student t test.
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