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Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model

Abstract

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a ‘differential multiple cleavage site’ model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.

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Acknowledgements

We are grateful to Dr Alexander Marcillo for assistance with surgical procedures. We are indebted to Brendan Battersby and Eric A Shoubridge for the NZB/BALB heteroplasmic mice and to New England Biolabs for the ScaI construct. We also thank the University of Miami Miller School of Medicine Imaging Core for their assistance. This work was supported by PHS grants EY10804 and NS041777. SB is supported by a supplement to PHS Grant EY10804.

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Correspondence to C T Moraes.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Bacman, S., Williams, S., Hernandez, D. et al. Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model. Gene Ther 14, 1309–1318 (2007). https://doi.org/10.1038/sj.gt.3302981

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