Abstract
In the first section of the thesis a novel gene transfer vector was designed to improve gene transfer efficiency as compared with standard nonviral gene vectors. Nonviral gene transfer still remains inefficient due to basic mechanistic barriers such as inefficient endosomal escape and, in particular, poor nuclear uptake of the DNA complexes. A novel promising strategy was developed to overcome these barriers by designing a peptide vector based on the arginine-rich motif of the HIV-1 TAT protein (TAT-peptide). The TAT-peptide has been shown to function as a protein transduction domain (PTD), i.e. to mediate translocation of heterologous proteins into cells by crossing cell membranes in a manner different from endocytosis. In addition, the TAT-peptide has been shown to function as nuclear localization sequence (NLS) which directly interacts with a nucleocytoplasmic shuttle protein to mediate nuclear import. As a consequence, these properties of the TAT-peptide should substantially improve gene delivery. In order to take the impact of polyelectrolyte binding and stability on gene transfer into consideration for vector design, the dimer, trimer and tetramer of the TAT-peptide was synthesized as novel peptide vectors and their properties for the use in gene transfer was investigated. It was attempted to find a correlation between the degree of oligomerisation of the TAT-peptide, the resulting biophysical parameters and the gene delivery efficiency in context with the unique cell physiologic functions of the TAT-peptide. In the second section of the thesis the gene transfer efficiency mediated by cationic polymers such as polyethylenimine (PEI) 25 kDa and fractured polyamidoamine dendrimers to the mouse lung in vivo was investigated. Previous studies have shown that the gene transfer efficiency mediated by PEI and fractured polyamidoamine dendrimers on bronchial epithelial cells was high even in the presence of lung surfactant. However, at this time no data were available for their potential to mediate gene transfer in vivo to the mouse lung. To investigate their gene transfer efficiency in vivo an intratracheal application method was developed. Gene transfer efficiency of both cationic polymers was optimised and the effect of lung surfactant on gene transfer efficiency was examined. In addition, the time course of transgene expression and the inflammatory response induced by the gene vectors was investigated. Gene transfer efficiency mediated by the cationic polymers was compared with the efficiency mediated by cationic lipids. In previous studies ligand modified gene vectors have been successfully used for receptor-mediated gene transfer. For this reason, this opportunity has been investigated for gene delivery to the lung. In addition, in this section the potential of the TAT-peptides to enhance gene transfer in the lung in vivo was examined.
Aerosol drug delivery currently represents the most acceptable and convenient delivery system for repeated drug application to the lungs. The group of Densmore has shown that PEI polyplexes can mediate gene transfer after nebulisation in vivo. However, gene transfer efficiency of PEI polyplexes has been shown to be influenced by the solvent used for complex formulation. In this context, the effect of jet nebulisation on such particle parameters of PEI/DNA polyplexes when formulated with commonly used solvents was investigated. The effect of jet nebulisation on particle parameters was compared to transfection efficiency in order to correlate physical parameters of the polyplexes with biological activity. |
