Gluconoylated and Glycosylated Polylysines As Vectors for Gene Transfer into Cystic Fibrosis Airway Epithelial Cells

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Abstract
To provide an alternative to viral vectors for the transfer of genes into airway epithelial cells in cystic fibrosis (CF), a novel set of substituted polylysines were employed. Polylysine was partially neutralized by blocking a number of positively charged residues with gluconoyl groups. In addition, polylysine was substituted with sugar residues on a specified number of amino groups. Using the gluconoylated polylysine as vector, the pCMVLuc plasmid gave high expression of the reporter gene luciferase in immortalized CF/T43 cells. The luciferase activity was 75-fold greater in the presence of 100 μM chloroquine. Luciferase gene expression persisted at high levels for up to at least 120 hr following transfection. Glycosylated polylysines/pCMVLuc complexes were compared to the gluconoylated polylysine/pCMVLuc complex and β-Gal-, α-Glc-, and Lac-substituted polylysines gave 320%, 300%, and 290%, respectively, higher expression of the reporter gene luciferase. Luciferase expression ranged from 35 to 2 ng of luciferase per milligram of cell protein in the order: β-Gal = α-Glc = Lac > α-Gal = Rha = Man > β-GalNAc > α-GalNAc = α-Fuc, suggesting that the transfection efficiency is sugar dependent. Most importantly, in primary cultures of both CF and non-CF airway epithelial cells grown from tracheal tissue explants, lactosylated polylysine gave uniformly high expression of luciferase. The glycosylated polylysines provide an attractive nonviral approach for the transfer of genes into airway epithelial cells. Carbohydrate-substituted polylysines have been shown to be highly effective vectors for the transfer of the reporter gene luciferase into cystic fibrosis (CF) airway epithelial cells in culture. Gluconoylated polylysine was also an effective vector in immortalized CF cells. However, with several of the glycosylated polylysines as vectors, the expression of luciferase was increased by three-fold. CF and non-CF cells in primary cultures showed a preference for lactose-substituted polylysine over the other glycosylated polylysines. Optimal conditions were examined regarding vector/plasmid ratio, time of transfection, days of reporter gene expression, concentration of chloroquine, and cell number. These novel vectors provide an attractive alternative to viral vectors for gene transfer into airway epithelial cells.