Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease

Abstract

Frequently, mtDNA with pathogenic mutations coexist with wildtype genomes (mtDNA heteroplasmy). Mitochondrial dysfunction and disease ensue only when the proportion of mutated mtDNAs is high, thus a reduction in this proportion should provide an effective therapy for these disorders. We developed a system to decrease specific mtDNA haplotypes by expressing a mitochondrially targeted restriction endonuclease, ApaLI, in cells of heteroplasmic mice. These mice have two mtDNA haplotypes, of which only one contains an ApaLI site. After transfection of cultured hepatocytes with mitochondrially targeted ApaLI, we found a rapid, directional, and complete shift in mtDNA heteroplasmy (2-6 h). We tested the efficacy of this approach in vivo, by using recombinant viral vectors expressing the mitochondrially targeted ApaLI. We observed a significant shift in mtDNA heteroplasmy in muscle and brain transduced with recombinant viruses. This strategy could prevent disease onset or reverse clinical symptoms in patients harboring certain heteroplasmic pathogenic mutations in mtDNA.gene therapy ͉ mitochondria

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

“…However, human cells lack a mechanism for import into mitochondria of cytoplasmically synthesized RNAs, and allotopic (nuclear) expression of wild-type counterparts of mitochondrially encoded components of the electron transport chain has met with only limited success, 25,26 presumably due to the severe limitations imposed on mitochondrial import of highly hydrophobic polypeptides. 26,27 Although the notion that many pathogenic mtDNA mutations are heteroplasmic and thus targeted elimination of mutant mtDNA can correct certain mitochondrial diseases has been suggested previously, 14,28,29 it has not been tested with pathogenic mtDNA mutations 28,29 or with a relevant gene delivery system. 14,28 Therefore, this study represents the first attempt to use a gene therapy approach for the selective destruction of mtDNA molecules carrying a pathogenic mutation.…”

Unsolved issues related to human mitochondrial diseases

“…Lowering even partially the mutation load could therefore significantly improve the phenotype. Several attempts have been done towards that direction either targeting the replication of the mutant mitochondrial DNA in cellular or animal models [18,19] or selecting for cells with lower mutation load in cellular models and in patients [20][21][22]. Although establishing the proof of principle these attempts have yet too show their efficacy on a broader field.…”

Transmitochondrial Mice as Models for Mitochondrial DNA-Based Diseases

“…In the case of patients who have already contracted mitochondrial diseases, however, it is not possible to apply this nuclear transplantation therapy. In in vitro studies using human cell models carrying pathogenic mutant mtDNAs, unique concepts by which mutant mtDNA molecules are digested by a mitochondrial-targeted restriction enzyme or zinc-finger nuclease have been proposed as a treatment strategy [2,25]. It has also been reported that ketogenic treatment is effective for heteroplasmic shift to lower levels of ∆mtDNA in human cells [34].…”