Gene editing of the wild-type APOE3 gene to the dysfunctional variants APOE2 or APOE4 using synthetic RNA-DNA oligonucleotides (chimeraplasts)

摘要

Plasma apolipoprotein E (apoE) is secreted by liver (>90%) and macrophages, and protects against atherosclerosis by contributing to cholesterol homeostasis and by locally restricting lesion development. Three common isoforms arise from single nucleotide polymorphisms (SNPs): apoE2, the rarest variant, differs from wild-type apoE3 by an R158C substitution and causes recessive Type III hyperlipidaemia, whereas apoE4 (C112R) produces a dominant hyperlipidaemia. Molecular explanations for these relationships are poorly defined, but most likely reflect differences in receptor binding and/or intracellular trafficking. Potentially, single-base mutations can be corrected in genes by using synthetic RNA-DNA oligonucleotides (chimeraplasts), which harness cellular mismatch repair mechanisms. Here, we evaluate gene editing of the APOE3 gene in HepG2 hepatoblastoma cells and THP-1 monocyte-macrophages, with the aim of producing new human clonal cell lines that secrete apoE2 or apoE4 for subsequent biological investigations. Initially, we used polyethyleneimine (PEI) to transfer chimeraplasts and showed that brief centrifugation improved conversion efficiency. A dose-dependent conversion was seen by PCR- RFLP analysis in HepG2 cells and confirmed by direct sequencing. Additional increases in conversion efficiency were also noted when receptor-dependent uptake was exploited, using galactotetraose-PEI and mannose-PEI for HepG2 and THP-1 cells, respectively. Unexpectedly, however, the conversions appeared unstable as analysing 50 clones isolated by limiting dilution from chimeraplast-treated THP-1 and HepG2 cells consistently revealed no genotypic changes. Whether this instability is due to cytotoxic or apoptotic effects of the transfection complex, or to a recent suggestion that cells have effective defence mechanisms that counteract targeted genome sequence alterations, remains to be established.