Lecithin-cholesterol acyltransferase (LCAT) is a high-density lipoprotein (HDL)-associated enzyme, which is secreted mainly by the liver. By esterifying cholesterol in the surface of immature HDL, LCAT drives reverse cholesterol transport, an important process in preventing atherosclerosis. Specific point mutations in the LCAT gene, producing serine to alanine amino acid substitutions at positions 208 or 216, are reported to increase enzymatic activity up to 14 times. Here, I attempt to create these mutations using a new technology, termed chimeraplasty targeted gene repair in situ using synthetic RNA-DNA oligonucleotides (chimeraplasts). First, I demonstrated that the Ser208Ala and Ser216Ala mutations do increase LCAT specific activity by comparing recombinant Ghinese hamster ovary (CHO) cells secreting wild-type LCAT (CHO-LCAT), LCATser2i6Aia, or LCATser208Aia+ser2i6Aia. I then targeted CHO-LCAT cells, and a human hepatoma cell line (HepG2), in vitro with chimeraplasts directed at the Ser208 and Ser216 sites. However, I was unable to create the required mymidine to guanine nucleotide substitution required using standard procedures, even by varying transfection conditions, repeat targeting, or altering chimeraplast design. I studied, therefore, chimeraplast uptake into the nucleus with various polyethylemmine (PEI)-based transfection reagents by using fluorescently-labelled oligonucleotides and a validated chimeraplast, able to mutate the apolipoprotein E (APOE) gene. I found that melittin-PEI, transferrin-PEI and galactose4-PEI were superior to linear PEI, but targeting the LCAT gene with these optimal reagents failed to produce either Ser208Ala or Ser216Ala mutations. Moreover, co-targeting cells simultaneously with LCAT and apoE chimeraplasts mutated the APOE gene, but not the LCAT gene. Finally, to investigate possible gene position or sequence effects I produced recombinant CHO cells expressing both LCAT and apoE targeting regions adjacent to each other. When these cells were co-targeted the Ser216Ala mutation was successful. I conclude that chimeraplast-directed gene mutation/ repair is a promising technique but further investigation is required to explain inconsistent results when targeting different cell lines.

Cardiovascular disease is one of the leading causes of death in the Western world. One of the most common causes is the rupture of unstable atherosclerotic plaques, which can lead to thrombus formation, occlusion of the artery and myocardial infarction. Therefore there is a need for treatments that stabilise vulnerable plaques. Gene therapy has the potential to provide a novel treatment for this. To maximise therapeutic gene expression and minimize any potential adverse effects due to unwanted transgene expression in non-target tissues, a gene delivery vector specifically targeted to areas of atherosclerotic vasculature is required. The vector is also required to efficiently infect cells to produce relatively long term transgene expression, be stable in blood, non-toxic, non-immunogenic and producible at high titres. Viral vectors, particularly those based on adenovirus (Ad) and adeno-associated virus (AAV) have many of the desired features, but transduce vascular cells relatively inefficiently and in a non-selective manner. Methods of altering their tropism have been established and could be utilised to develop vectors with a high degree of selectivity for atherosclerotic plaques. Detargeting of vectors can be achieved by mutating regions of the virus capsid that are thought to bind the native cellular receptors and retargeting to novel cell types is achieved by inserting peptide ligands into the virus capsid. The aim of this study was to develop atherosclerotic plaque targeted vectors and to characterise Ad and AAV vector platforms in this regard. Two approaches were taken. In the first approach three previously identified plaque targeting peptides were tested for their ability to target viral vectors to atherosclerotic plaques. The second approach involved performing phage display in a mouse model of plaque rupture to identify novel peptides that specifically target unstable plaques. Further work was carried out to characterise and develop methods for using an AAV2 based peptide library as a novel tool for biopanning. This work has provided further characterisation of Ad and AAV platform vectors that may be utilised in the development of vectors with a highly selective tropism.