Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease

Abstract

Mutations in the mitochondrial DNA (mtDNA) can cause a variety of human diseases. In most cases, such mutations are heteroplasmic (i.e. mutated and wild-type mtDNA coexist) and a small percentage of wild-type sequences can have a strong protective effect against a metabolic defect. Because a genetic approach to correct mtDNA mutations is not currently available, the ability to modulate heteroplasmy would have a major impact in the phenotype of many patients with mitochondrial disorders. We show here that a restriction endonuclease targeted to mitochondria has this ability. A mitochondrially targeted PstI degraded mtDNA harboring PstI sites, in some cases leading to a complete loss of mitochondrial genomes. Recombination between DNA ends released by PstI was not observed. When expressed in a heteroplasmic rodent cell line, containing one mtDNA haplotype with two sites for PstI and another haplotype having none, the mitochondrial PstI caused a significant shift in heteroplasmy, with an accumulation of the mtDNA haplotype lacking PstI sites. These experiments provide proof of the principle that restriction endonucleases are feasible tools for genetic therapy of a sub-group of mitochondrial disorders. Although this approach is limited by the presence of mutation-specific restriction sites, patients with neuropathy, ataxia and retinitis pigmentosa (NARP) could benefit from it, as the T8399G mutation creates a unique restriction site that is not present in wild-type human mitochondrial DNA.

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.…”

“…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.…”

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

“…The same construct was able to shift mtDNA heteroplasmy in cultured cells containing both mouse and rat mtDNA, because the latter lacks PstI sites (8). In a similar study, targeting the restriction endonuclease SmaI to mitochondria of cultured cells harboring the T8399G NARP mutation prompted specific elimination of mutated mtDNA, followed by repopulation by the wild-type mtDNA.…”

Mitochondrial DNA mutations in human cancer

“…Targeted restriction endonucleases have been used as a tool for treatment of mitochondrial dysfunction, The SmaI gene with a mitochondrial targeting sequence, when expressed in cybrids carrying mutant mtDNA, resulted in elimination of the mutant mtDNA, followed by progression of the wild-type mtDNA, which led to the restoration of normal intracellular ATP level and normal mitochondrial membrane potential (Tanaka et al, 2002). Earlier studies have also demonstrated the targeting of endonucleases (PstI) to mitochondria as a potential tool for mitochondrial gene therapy (Srivastava and Moraes, 2001). Other therapeutic approaches included the use of Oligomycin which showed an increase of wild-type cells over cells harboring the mutation T8993G of mtATP6 gene (Manfredi et al, 1999).…”