Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids
Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids
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DOI：
10.1038/ng0297-212
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发表时间：
1997-02-01
期刊：
影响因子：
30.8
通讯作者：
Lightowlers, RN
中科院分区：
文献类型：
--
作者：
Taylor, RW;Chinnery, PF;Lightowlers, RN
线粒体DNA （mtDNA）是人类唯一的染色体外DNA。它是一个小的（16.5 kb）基因组，编码呼吸链的13个必需肽，2个rnas和22个tRNAs。这种基因组的缺陷现在被认为是疾病的重要原因，可能以点突变或重排的形式出现（1-3）。对于mtDNA突变的患者没有有效的治疗方法(4)。在大多数mtDNA缺陷患者中，突变型和野生型分子同时存在于同一个细胞中，这种现象被称为细胞内异质性。此外，在异质性存在的情况下，有一个阈值，在疾病变得生化和临床明显之前，一定水平的突变mtDNA是必要的（5-7）。基于异质性的存在和这些突变的隐性性质，我们相信有可能通过选择性抑制突变mtDNA的复制来治疗患者，从而只允许野生型分子的繁殖。为了证实这种方法的有效性，我们合成了与含有缺失断点或单碱基突变的人类mtDNA模板互补的肽核酸（PNAs），这两种突变都有证据表明会导致疾病。利用生理条件下的体外复制径流试验，抗基因组PNAs特异性地抑制突变型mtDNA模板的复制，而不是野生型mtDNA模板。此外，我们已经证明这些PNAs被吸收到培养的人成肌细胞中。因此，我们相信我们已经确定了抗基因组PNA治疗异质mtDNA疾病患者的潜在价值。
Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in humans. It is a small (16.5 kb) genome which encodes 13 essential peptides of the respiratory chain, two rRNAs and 22 tRNAs. Defects of this genome are now recognized as important causes of disease and may take the form of point mutations or rearrangements(1-3). There is no effective treatment for patients with mtDNA mutations(4). In the majority of patients with mtDNA defects, both mutant and wild-type molecules are present in the same cell - a phenomenon known as intracellular heteroplasmy. In addition, in the presence of heteroplasmy there is a threshold whereby a certain level of mutant mtDNA is necessary before the disease becomes biochemically and clinically apparent(5-7) Based on the presence of heteroplasmy and the recessive nature of these mutations, we believe it will be possible to treat patients by selectively inhibiting the replication of the mutant mtDNA, thereby allowing propagation of only the wild-type molecule. To confirm the validity of such an approach we synthesised peptide nucleic acids (PNAs) complementary to human mtDNA templates containing a deletion breakpoint or single base mutation, both mutations well documented to cause disease. Using an in vitro replication run-off assay under physiological conditions, the antigenomic PNAs specifically inhibited replication of mutant but not wild-type mtDNA templates. Furthermore, we have shown uptake of these PNAs into cultured human myoblasts. We believe that we have therefore established the potential value of antigenomic PNA therapy for patients with heteroplasmic mtDNA disorders.