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
