Disterer, P.; (2008) Oligonucleotide-mediated gene editing of Apolipoprotein A-I. Doctoral thesis , University of London.

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Abstract

Apolipoprotein A-I (ApoA-I) is the major protein constituent of high density lipoprotein (HDL) and controls reverse cholesterol transport, an important process in preventing atherosclerosis. A natural point mutation, ApoA-lMiiano (ApoA-Im) enhances the atheroprotective potential of HDL. Here, I attempt to introduce this specific modification into the genome of mammalian cells using the gene therapy strategy of oligonucleotide-mediated gene editing. I showed successful APOA-I gene editing in recombinant Chinese hamster ovary (CHO-AI) and human hepatocellular carcinoma (HepG2) cells by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing. However, I was unable to isolate gene-edited cell clones or quantify gene editing. Therefore, I established a recombinant CHO cell line expressing mutated non-fluorescent enhanced green fluorescent protein (mEGFP) that demonstrates successful gene editing through restoration of fluorescence. Using flow cytometry, I studied the influence of transfection reagents and oligonucleotide design on gene editing efficiency and viability of gene-edited cells. I found that Lipofectamine 2000 generated higher initial editing efficiencies with phosphorothioate (PTO) or locked nucleic acid (LNA) modifications, but unmodified oligonucleotides produced significantly more gene-edited clones. I also investigated ways of increasing editing efficiencies or selecting for gene-edited cells independent of the target gene and demonstrated that I had introduced three specific nucleotide alterations by Southern blotting of genomic DNA from gene-edited cell clones. I established that the phenotype in these clones was unstable due to epigenetic down- regulation which was not specific to the gene-edited allele. Variegated gene expression in three mEGFP cell lines demonstrated that gene editing was positively associated with target gene expression. Isolation of ApoA-lM gene-edited cells using flow cytometry failed because an ApoA-I i homodimer antibody was not specific. In summary, I demonstrated that oligonucleotide-mediated gene editing produces stable gene-edited cells in a reporter gene system. However, further research is needed before this method can be applied to non-selectable genes.

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