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S. R. Bacman, S. L. Williams, D. Hernandez, C. T. Moraes; Manipulating Heteroplasmy by Delivering Restriction Endonucleases to Muscle Mitochondria in a "Differential Multiple Cleavage-Site" Model. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3003.
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Extraocular muscles have high mitochondrial content and elevated energy demands and consequently are primarily involved in mitochondrial diseases such as Chronic Progressive External Ophthalmoplegia (CPEO), a condition commonly caused by heteroplasmic mtDNA mutations. By lowering the load of mutant mtDNA one could eliminate the disease phenotype. Because single-site mitochondrially-targeted restriction endonucleases (RE) were shown to modify mtDNA heteroplasmy, we tested if this approach could be applied in a multiple-site condition, where a mtDNA mutation creates an extra site to the multiple sites originally present in the wild-type mtDNA
Taking advantage of a mouse model harboring two different mtDNA haplotypes (BALB/NZB) and using recombinant adenovirus as a gene vector, we delivered a mitochondrially-targeted ScaI restriction endonuclease with an HA tag (rAV-Mito-Sca-HA) to skeletal muscle. ScaI recognizes both mtDNA haplotypes differentially (5 sites in NZB and 3 sites in BALB mtDNA). The rAV was injected intramuscularly in the right quadriceps of 5 days old mice. Muscle samples were analyzed weekly (up to 6 weeks) for shifts in mtDNA heteroplasmy (determined by last cycle hot PCR/RLFP).
Our results showed that expression of the recombinant rAV-Mito-Sca-HA could be detected in muscle up to 4 weeks after injections. We observed small but significant changes in mtDNA heteroplasmy in the predicted direction (decreasing the 5-site NZB mtDNA/3-site-BALB mtDNA ratio by approximately 15-20%) in injected areas. These included both cytochrome oxidase (COX) deficient fibers areas and COX-positive fibers adjacent to COX-deficient areas.
Although a cytochrome oxidase deficiency, likely caused by mtDNA depletion, was observed in some muscle fibers, our data suggest that, when carefully controlled, the expression of Mito-RE in the context of multiple cleavage-sites has the potential to modulate mtDNA heteroplasmy in mitochondrial diseases.
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