Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Minczuk, Michal; Papworth, Monika; Kolasinska, Paulina; Murphy, Michael P.; Klug, A.

doi:10.1073/pnas.0609502103

Cited by 143 publications

(114 citation statements)

References 33 publications

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“…In this regard, a recent study has demonstrated the feasibility of sequence-specific modification of mtDNA with a chimeric zinc-finger methylase, 30 which suggests a solution to both of the above problems.…”

Section: Discussionmentioning

confidence: 99%

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

et al. 2008

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Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a timeand dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

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Section: Discussionmentioning

confidence: 99%

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

et al. 2008

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“…21 Methyltransferases fused to zinc finger proteins targeting predetermined sites in the DNA to repress gene expression have been reported. 12,22,23 Engineering ZFPs targeted to the EpCAM promoter 21 fused to M.SssI as an effector domain, provides a powerful tool to achieve targeted methylation. The ZFP binds specific to the promoter where after the enzyme will methylate only the CpGs close to the ZFP target sequence.…”

Section: Discussionmentioning

confidence: 99%

Persistent downregulation of the pancarcinoma‐associated epithelial cell adhesion molecule via active intranuclear methylation

Gun

Wasserkort

Monami

et al. 2008

Intl Journal of Cancer

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The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM-negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAMpositive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM-positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA-mediated silencing remained for 4 days, after which EpCAM re-expression increased in time, while M.SssI-mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.

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“…The possibility of selective targeting of mtDNA has also been explored by Minczuk et al 19 These authors have attempted to use the short zinc-finger peptides that show sequence-specific DNA binding capabilities.…”

Section: Manipulating the Mitochondrial Genome May Lead To Rescue Of mentioning

confidence: 99%

Progress and prospects: gene therapy for mitochondrial DNA disease

et al. 2008

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Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase

Cited by 143 publications

References 33 publications

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes

Persistent downregulation of the pancarcinoma‐associated epithelial cell adhesion molecule via active intranuclear methylation

Progress and prospects: gene therapy for mitochondrial DNA disease

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