Abstract
The aim of gene therapy is to treat, cure or prevent a disease by replacing defective genes, introducing new genes or changing the expression of a person’s genes. Success of gene therapy is dependent on successful delivery of DNA from the site of administration into cell nuclei. Naturally occurring and
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highly efficient gene delivery systems are viruses. However, issues of safety, limited targeting possibilities and limited loading capacity justify the search for safe and flexible synthetic (non-viral) alternatives. Despite numerous efforts, success of non-viral gene delivery is hampered due to four major problems: toxicity, short-lived expression, heterogeneity of expression and low efficiency. The aim of this thesis is to rationally design DNA-Transporting Nanoparticles (DTN), focusing on three levels: (1) design of DNA to increase safety, efficiency and duration of transgene expression, (2) design of formulations to increase efficiency of transfection and (3) development of methods to allow better analysis of DTN. First it is shown how rational design of plasmid DNA can increase safety, efficiency and duration of transgene expression. Next, two strategies to improve nuclear uptake of DTN are examined. The first strategy involves modification of plasmid with DNA nuclear Targeting Sequences (DTS) which would facilitate nuclear localization by inducing coating of plasmid DNA with nuclear localization signals (NLS)-containing proteins and subsequent binding to importins. Modification of plasmids with DTS was not shown to increase gene expression, regardless of the cell line, dose and carrier system chosen, indicating that a bottleneck exists downstream of nuclear delivery. The second strategy was based on the non-covalent coupling of NLS-peptides to DNA. Peptides were shown to enhance transfection efficiency, but in an NLS-independent way. Next, the number of plasmids per DTN is addressed. The amount of active DNA could be reduced substantially while maintaining transfection activity by replacing active DNA with non-coding junk DNA. The results indicate that not the total amount of DNA, but the number of active DNA-containing particles is the critical factor in determining transfection efficiency. Size of DTN is a critical parameter, but preparation of DTN generally yields heterogeneously sized populations. Additionally, the size of DTN is affected by exposure to salts and proteins. A new method based on flow cytometry was developed to measure particle size distributions directly in biological fluids. Simultaneous detection of fluorescence and side scattering intensity (SSC ; indicative of size) provided the possibility to distinguish fluorescently labeled nanoparticles of interest from other particulate matter frequently present in biological fluid. Additionally, a proof of principle to sort heterogeneous populations into different size fractions was obtained. Testing of new gene delivery reagents is usually performed by reading out transgene expression levels relative to a reference formulation after in vitro transfection. A large number of variables that affect transfection efficiency was investigated. Based on this dataset, a screening protocol is suggested with the aim of standardization within the field. The research described in this thesis contributes to the understanding of the possibilities and limitations for design and testing of DNA-Transporting Nanoparticles for non-viral gene delivery.
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