期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 103, 期 52, 页码 19689-19694出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0609502103
关键词
gene therapy; mitochondria; mitochondrial diseases; synthetic zinc finger peptides; nuclear export signal
资金
- Medical Research Council [MC_U105184324, MC_U105663142] Funding Source: Medline
- MRC [MC_U105663142, MC_U105184324] Funding Source: UKRI
- Medical Research Council [MC_U105184324, MC_U105663142] Funding Source: researchfish
We used engineered zinc finger pepticles (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a UP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.
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