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Carlos T. Moraes, Sandra R. Bacman, Sion L. Williams; Gene Therapy for Mitochondrial Diseases with Mitochondria-Targeted Restriction Endonucleases. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4377.
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Mutations in mtDNA are often heteroplasmic and very high percentages of a mutant allele are necessary for clinical manifestations. We developed an approach to shift heteroplasmy levels using mitochondria-targeted restriction endonucleases (RE) to promote double strand breaks in specific mtDNA haplotypes. We investigated the effectiveness of systemic delivery (IP injection of mitochondria-targeted RE) in neonatal mice.
As a model of mtDNA heteroplasmy we used mice from a heteroplasmic strain harboring BALB and NZB mtDNA haplotypes. BALB mtDNA has one ApaLI restriction site and NZB mtDNA has none. The gene coding for ApaLI restriction endonuclease was cloned in an AAV9 backbone with a hemagglutinin (HA) tag and viral particles produced (AAV9-ApaLI-HA). Approximately 5x1011 viral genomes were injected intraperitoneally (IP) in 2-3 day-old mice. AAV9-Alkaline Phosphatase (AAV9-AP) was used as a control. Mice were euthanized at 6 weeks or 3 months post-injection.
Cardiac and skeletal muscle expression of the control rAAV9[AP] (alkaline phosphatase ) was observed following IP injection of rAAV9 at 6 weeks and 3 months post-injection. Liver showed limited focal positive staining. Other tissues including kidney, spleen, brain and lung showed no expression of AP at any time point. Expression of AAV9[mito-ApaLI-HA] was also confirmed in cardiac and skeletal tissues by western blotting. AAV9[mito-ApaLI-HA] induced a significant increase in NZB mtDNA in cardiac tissue and skeletal tissues (e.g. soleus, tibialis-anterior, gastrocnemius, quadriceps and EDL), at 6 weeks and 3 months post-injection. No significant change in NZB mtDNA was seen in kidney, spleen, brain and lung.
Mitochondria-targeted restriction endonucleases (RE) can eliminate target mtDNA haplotypes in specific tissues following systemic delivery in neonates. The current work underscores the therapeutic potential of mitochondrial nucleases using viral vectors with specific organ tropism.
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