The extra- and intracellular barriers to lipid and adenovirus-mediated pulmonary gene transfer in native sheep airway epithelium

Abstract

Gene transfer to the respiratory epithelium is currently apical membrane represented a significant barrier to both suboptimal and may be helped by the identification of limitagents. Adenovirus-mediated expression could be signifiing biological barriers. We have, therefore, developed an cantly augmented by increasing contact time or by preex vivo model which retains many of the characteristics of treatment of tissues with a nominally calcium-free medium. in vivo native airways including mucociliary clearance,The presence of these extracellular and plasma membrane mucus coverage and an intact cellular structure. Using this barriers appeared to be the key parameters responsible for model we have demonstrated several barriers to gene the approximately three log difference in gene expression transfer. Liposome-mediated gene transfer was inhibited found in vitro compared with our ex vivo model. Cytoskeleby normal mucus, with removal of this layer increasing tal elements and the cell cycle also influenced in vitro gene expression approximately 25-fold. In addition both lipotransfer, and represent further barriers which need to be some and adenovirus were inhibited by CF sputum. The overcome.

Poly (D, L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged transgene expression in airway epithelium in vitro, ex vivo and in vivo

“…Concomitant downregulation of infection and inflammation may in itself improve transfection efficiency by decreasing some of the extracellular barriers to airway gene transfer. 16 A number of potential disadvantages of PLG microspheres also emerged from this study. Gene transfer efficiency, particularly in vivo, is likely too low for clinical efficacy, even though the vexed question of the level of gene expression needed to achieve clinical benefit in CF remains unresolved.…”

“…16 Transgene expression was assayed for up to 96 h and again compared with lipid-#67-mediated gene transfer. …”

“…Sheep trachea 16 2 ) and transported to the laboratory on ice. To remove the mucus, the noncartilaginous sections were cut longitudinally to leave horse-shoe-shaped segments that were placed in 10 mm Petri dishes containing MEM with 50 mg/ml gentamycin and 1 Â antibiotic/antimycotic solution (MEM-A) with the tips of the cartilage submerged in the media.…”

“…15 pDNA encapsulated in PLG remains stable, and protected from enzymatic degradation. 5,16 In this study, we have explored the potential use of PLG microspheres for sustained gene delivery and expression in airway epithelium, with a view to further application for cystic fibrosis gene therapy. Following formulation and characterization of the microspheres, gene transfer efficiency and duration of transgene expression have been tested in vitro on both non-CF and CF cell lines, ex vivo in a model using native sheep trachea and finally in vivo by instillation into the lungs of Balb/C mice.…”

Gene therapy for barrett's esophagus: adenoviral gene transfer in different intestinal models

“…NAC is a mucolytic agent, which exhibited an improved gene transfer in pulmonary epithelium for both viral and nonviral vectors. 25,26 Studies on the effect of mucus and the role of NAC in gene transfer of the gastrointestinal tract are limited. An earlier study showed that NAC removed the mucus between the villi, but no attempt of gene transfer was made.…”