Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease

Abstract

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double strand breaks (DSB) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In the present work we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results demonstrate the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Emerging therapeutic approaches to mitochondrial diseases

“…T8993G is associated with neuropathy, ataxia, and retinitis pigmentosa (NARP), or when present at higher levels (>90%), with a more severe maternally inherited syndrome (MILS) [DiMauro and Schon, 2003].Organ-specific shifts in mtDNA heteroplasmy following intravenous systemic delivery of a mitochondriatargeted ApaLI-RE was shown in heart and liver using cardiotropic AAV6 and hepatotropic adenovirus as viral vectors [Bacman et al, 2010]. As the mitoApaLI only cleaves BALB mtDNA, and the remaining NZB mtDNA replicates to maintain normal mtDNA copy numbers, no significant mtDNA depletion was observed [Bacman et al, 2010].…”

Mitochondrial Genetic Manipulation

“…Protein genes normally carried by the mtDNA (encoding oxidative phosphorylation chain subunits, CMS polypeptides) and genes encoding heterologous proteins, restriction endonucleases, or specific zinc-finger proteins were expressed in the nucleus after addition of a sequence coding for a mitochondrialtargeting peptide; the mRNAs were either a translated on free cytosolic polysomes or b specifically targeted to the mitochondrial surface, leading to membrane-bound polysomes; translocation into the organelles was through the regular protein import pathway. mRNA messenger RNA, mtDNA mitochondrial DNA, CMS cytoplasmic male sterility mitochondrial targeting of appropriate restriction enzymes in mammalian cell lines and animal models (Bayona-Bafaluy et al 2005;Alexeyev et al 2008;Bacman et al 2010;). In each case, an efficient shift in the mtDNA heteroplasmy was obtained.…”

Pathologies de l’ADN mitochondrial et stratégies thérapeutiques

“…Une augmentation significative de la consommation d'oxygène est observée dans les cellules ainsi traitées. Il est également possible de cibler spécifiquement un organe via l'utilisation de vecteurs adénoviraux AAV (adeno-associated virus), tels que des vecteurs cardiotropiques et hépatotropiques qui, chez la souris, ont permis de délivrer l'enzyme ApaLI dans le coeur et le foie, et d'induire une diminution du niveau d'hétéroplasmie [26].…”