Cellular processing and nuclear targeting of non-viral gene delivery systems

Abstract

Gene therapy utilizes genetic material in order to cure patients either by DNA vaccines or by replacement of a defective gene with a normal one. For successful gene therapy certain elements are required: gene delivery systems with low toxicity and immunity, with efficient gene transfer and high gene expression in the targeted cells or tissue at a functional level and, preferably, in a controllable manner.

Based on the type of gene delivery vehicle, a distinction in gene therapy strategies can be made between viral and non-viral delivery systems.

Viral vectors are derived from viruses where genetic elements of the virus are replaced by therapeutic genes. Non-viral gene therapy offers a saver alternative for viral gene therapy. Non-viral gene therapy is based on the introduction of plasmid DNA into the cell by physical methods and cationic carriers.

This thesis deals with the intracellular trafficking of complexes consisting of cationic carriers, like polymers and liposomes, and plasmid DNA, designed for gene delivery. Different aspects of the cellular processing of these polymers have been assessed, like cellular binding and internalization, intracellular trafficking and nuclear import.

In COS-7 cells uptake of PEI- and pDMAEMA-based polyplexes was via both the clathrin- and caveolae-mediated route. However, only the latter route was important for expression of the reporter gene.

In an attempt to improve the efficiency of cationic polymers, cells were transfected with linear or plasmid DNA equipped with an NLS peptide. Both a classical and a non-classical NLS-peptide attached directly to the DNA did not improve the transfection efficiency of the complexes of DNA and cationic polymers. However, when the NLS peptides were coupled to the cationic polymer poly-L-lysine (PLL) prior to complexation of the DNA in small particles, the transfection efficiency of lipopolyplexes, consisting of plasmid DNA, NLS-PLL and cationic liposomes, was enhanced.

Taken together, these results show that successful import of DNA into the nucleus of non-dividing cells can be achieved by complexation of the DNA into small, i.e. smaller than 39 nm, particles and coupling of an NLS peptide to the polymer surface rather than direct attachment to the DNA.

In conclusion, the ultimate gene delivery system combines the efficiency advantage of viral gene therapy with the safety advantage of non-viral gene therapy.