Manzano Anna, Mohri Zahra, Sperber Galia, Ogris Manfred, Graham Ian, Dickson George, Owen James S
Department of Medicine, Royal Free and University College Medical School, London NW3 2PF, UK.
J Gene Med. 2003 Sep;5(9):795-802. doi: 10.1002/jgm.403.
BACKGROUND: Elevated plasma high-density lipoprotein (HDL), and its major constituent apolipoprotein AI (apoAI), are cardioprotective. Paradoxically, two natural variants of apoAI, termed apoAI(Milano) and apoAI(Paris), are associated with low HDL, but nevertheless provide remarkable protection against heart disease for heterozygous carriers and may even lead to longevity. Both variants arise from point mutations and have Arg(173) and Arg(151) to Cys substitutions, respectively, which allow disulphide-linked dimers to form. Potentially, synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct single point mutations in genomic DNA. Here, we use a variation of such targeted gene repair technology, 'gain-of-function chimeraplasty', and attempt to enhance the biological activity of apoAI by altering a single genomic base to generate the atheroprotective phenotypes, apoAI(Milano) and apoAI(Paris). METHODS: We targeted two cultured cell lines that secrete human apoAI, hepatoblastoma HepG2 cells and recombinant CHO-AI cells, using standard 68-mer chimeraplasts with polyethyleneimine (PEI) as carrier and then systematically varied several experimental conditions. As a positive control we targeted the dysfunctional APOE2 gene, which we have previously converted to wild-type APOE3. RESULTS: Conversion of wild-type apoAI to apoAI(Milano) proved refractory, with limited correction in CHO-AI cells only. However, a successful conversion to apoAI(Paris) was achieved, as demonstrated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct genomic sequencing. Unexpectedly, attempts with a new batch of 68-mer chimeraplast to enhance conversion, by using different delivery vehicles, including chemically modified PEI, failed to show a base change; nor could conversion be detected with an 80-mer or a 52-76-mer series. In contrast, when a co-culture of CHO-E2 and CHO-AI cells was co-targeted, a clear conversion of apoE2 to apoE3 was seen, whereas no apoAI(Paris) could be detected. When the individual chimeraplasts were analysed by denaturing electrophoresis only the active apoE2-to-E3 chimeraplast gave a sharp band. CONCLUSIONS: Our findings suggest that different batches of chimeraplasts have variable characteristics and that their quality may be a key factor for efficient targeting and/or base conversion. We conclude that, although an evolving technology with enormous potential, chimeraplast-directed gene repair remains problematical.
背景：血浆高密度脂蛋白（HDL）及其主要成分载脂蛋白AI（apoAI）水平升高具有心脏保护作用。矛盾的是，apoAI的两种天然变体，即apoAI（米兰）和apoAI（巴黎），与低HDL相关，但却能为杂合子携带者提供显著的心脏病保护作用，甚至可能延长寿命。这两种变体均由点突变产生，分别有精氨酸（173）和精氨酸（151）被半胱氨酸取代，从而形成二硫键连接的二聚体。合成的RNA/DNA寡核苷酸（嵌合体）有可能永久纠正基因组DNA中的单点突变。在此，我们使用这种靶向基因修复技术的一种变体——“功能获得性嵌合体技术”，试图通过改变单个基因组碱基来增强apoAI的生物活性，以产生具有抗动脉粥样硬化表型的apoAI（米兰）和apoAI（巴黎）。 方法：我们使用以聚乙烯亚胺（PEI）为载体的标准68聚体嵌合体靶向两种分泌人apoAI的培养细胞系，即肝癌细胞系HepG2细胞和重组CHO-AI细胞，然后系统地改变几个实验条件。作为阳性对照，我们靶向功能失调的APOE2基因，我们之前已将其转化为野生型APOE3。 结果：将野生型apoAI转化为apoAI（米兰）被证明难以实现，仅在CHO-AI细胞中有有限的纠正。然而，通过聚合酶链反应-限制性片段长度多态性（PCR-RFLP）分析和直接基因组测序证明成功转化为apoAI（巴黎）。出乎意料的是，尝试使用包括化学修饰的PEI在内的不同递送载体，用一批新的68聚体嵌合体来增强转化，未显示碱基变化；使用80聚体或52 - 76聚体系列也未检测到转化。相反，当共同靶向CHO-E2和CHO-AI细胞的共培养物时，可见apoE2明显转化为apoE3，而未检测到apoAI（巴黎）。当通过变性电泳分析单个嵌合体时，只有活性apoE2到E3嵌合体产生清晰的条带。 结论：我们的研究结果表明，不同批次的嵌合体具有可变特性，其质量可能是有效靶向和/或碱基转化的关键因素。我们得出结论，尽管嵌合体介导的基因修复是一项具有巨大潜力的不断发展的技术，但仍然存在问题。
Mol Reprod Dev. 2005-6
Oligonucleotides. 2011
Nucleic Acids Res. 2005-7-7
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