Effect of Host Modification and Age on Airway Epithelial Gene Transfer Mediated by a Murine Leukemia Virus-Derived Vector

Abstract

To study retroviral gene transfer to airway epithelia, we used a transient transfection technique to generate high titers (∼109 infectious units/ml after concentration) of murine leukemia virus (MuLV)-derived vectors pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G). Transformed (CFT1) and primary airway epithelial cells were efficiently transduced by a VSV-G-pseudotyped lacZ vector (HIT-LZ) in vitro. CFT1 cells and primary cystic fibrosis (CF) airway cell monolayers infected with a vector (HIT-LCFSN) containing human CF transmembrane conductance regulator (CFTR) in the absence of selection expressed CFTR, as assessed by Western blot analysis, and exhibited functional correction of CFTR-mediated Cl− secretion. In vitro studies of persistence suggested that pseudotransduction was not a significant problem with our vector preparations. In a sulfur dioxide (SO2) inhalational injury model, bromodeoxyuridine (BrdU) incorporation rates were measured and found to exceed 50% in SO2-injured murine tracheal epithelium. HIT-LZ vector (multiplicity of infection of ∼10) instilled into the SO2-injured tracheas of anesthetized mice transduced 6.1% ± 1.3% of superficial airway cells in tracheas of weanling mice (3 to 4 weeks old; n = 10), compared to 1.4 ± 0.9% in mice 5 weeks of age (n = 4) and 0.2% in mice older than 6 weeks (n = 15). No evidence for gene transfer following delivery of HIT-LZ to tracheas of either weanling or older mice not injured with SO2 was detected. Because only a small fraction of BrdU-labeled airway cells were transduced, we examined the stability of the vector. No significant loss of vector infectivity over intervals (2 h) paralleling those of in vivo protocols was detected in in vitro assays using CFT1 cells. In summary, high-titer vectors permitted complementation of defective CFTR-mediated Cl− transport in CF airway cells in vitro without selection and demonstrated that the age of the animal appeared to be a major factor affecting in vivo retroviral transduction efficiency.

Enhanced Epithelial Gene Transfer by Modulation of Tight Junctions with Sodium Caprate

“…Cultured CF HAE cells grown on 12-mm Transwell-Col inserts were mounted in modified Ussing chambers interfaced with an electrometer, in which transepithelial potential difference (V t ) and resistance were monitored continuously (13). Basal V t , resistance, and current were recorded, and the sequential effects of amiloride (10 Ϫ 4 M), luminal Cl Ϫ substitution, forskolin (10 Ϫ 5 M), and uridine triphosphate (10 Ϫ 5 M) on these parameters in Ad CFTRinfected and control cultures were measured.…”

The Status of Gene Therapy for Cystic Fibrosis

“…Administration of retrovirus vector to lungs of mice with sulfur dioxide-induced acute lung injury resulted in gene expression in only a small percentage of tracheal epithelial cells. 96 Lentivirus vectors also integrate and are more likely to result in sustained gene expression. 97,98 In contrast to conventional retroviruses, lentiviruses do not require cell division for entry and gene expression and have been pseudotyped with surface proteins that permit binding and entry to airway epithelium.…”

“…[97][98][99] Similar to adenoviruses, cellular barriers to viral binding and internalization appear relevant to lentiviruses because basolateral delivery appears necessary for gene transfer for viruses that are pseudotyped with vesicular stomatitis virus-G envelope. 91,96 Alternative envelope proteins are being developed that may overcome this limitation by facilitating entry via the apical membrane of polarized airway epithelium. Nonetheless, to date, levels of expression following airway administration of lentivirus vectors are low in animal models and there is little information about the gene transfer efficiency of lentiviruses in injured lung.…”

Airway Epithelia

“…Using this model, mice were exposed to SO 2 for 3 h at 500 ppm, and a HIV (VSV-G) vector was delivered onto the nasal or tracheal epithelia of rodents (8). Efficient reporter gene transfer was observed in nasal and tracheal airway epithelia, whereas no gene transfer was detected in the nasal or tracheal airway epithelia of sham (air)-exposed controls (8,12). Gene transfer to murine trachea was also more efficient following SO 2 exposure when vector was administered on the same day of exposure (~7% of cells transduced) and when cell proliferation was not increased, as compared to the day after SO 2 inhalation (2% transduction of cells) at which time peak airway cell proliferation occurs.…”