Glycosylated polylysines are novel, carbohydrate-based, gene transfer vectors, and have been shown to be effective and efficient for the transfer of reporter genes and the cystic fibrosis transmembrane conductance regulator (CFTR) gene into airway epithelial cells from patients with cystic fibrosis (CF). The long term goal is to develop glycosylated polylysines as vectors for the transfer of normal copies of the CFTR gene into the airways of CF patients. These novel polylysines have 30-40 percent of the epsilon-amino groups substituted by specific carbohydrates and thus can be customized to reflect the cell type. Lactosylated polylysine, with enhancing agents, gives 90 percent efficiency of transfection in CF cells, similar to that achieved by viruses. The lactosylated polylysine/DNA complex will be refined for experiments in vivo. These include optimizing the size of polylysine to be lactoxylated, the carbohydrate substitution and the solubility and size of the lactosylated polylysine/DNA complex. The receptor-mediated endocytosis into the CF airway cells will be examined and the fate of the complex will be followed within the cells using fluorescent labeled vector and a green fluorescent protein (GFP) plasmid. In timed studies with the confocal microscope, the arrival at the nucleus and the expression of GFP protein will be examined. The effect of enhancing agents on the integrity of the vector/plasmid complex will provide information on endosomal release during the transfection process. To demonstrate the adaptability of these glycosylated polylysines a number of specific human cell types will be examined with polylysine substituted by different carbohydrates reflecting identified specific surface receptors. The in vitro systems will serve to define the methods necessary for the in vivo studies which include transfer of the CFTR cDNA into animals. The most important in vivo model to be used will be the culture of human CF airway cells in a tracheal explant in a mouse xenograft model, to demonstrate a functional correction of the CF phenotype. In further in vivo experiments, transgenic CF mouse models will be used. The proposed studies will provide the information necessary to determine the role of these novel non-viral vectors in the gene therapy of CF. Moreover, they are directly applicable to other pulmonary diseases including inherited deficiencies of the surfactant proteins. If successful, they could provide the foundation for future clinical trials of gene transfer with these carbohydrate-based vectors. Due to the substitutions by different carbohydrates, the glycosylated polylysines may have a broad range of clinical applications including targeting to other tissues which are affected by a variety of other genetic diseases.
|Effective start/end date||6/15/99 → 5/31/01|
- National Institute of Diabetes and Digestive and Kidney Diseases: $175,000.00
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