title: 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.
authors.affiliation: Department of Cell Biology and Anatomy, Department of Neurology, University of Miami School of Medicine, 1095 NW 14th Terrace, Miami, FL 33136, USA.
authors.forename: S
authors.initials: S
authors.lastname: Srivastava
authors.forename: C T
authors.initials: CT
authors.lastname: Moraes
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D006239:Haplotypes; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000235:genetics* / Q000502:physiology; D051381:Rats
chemical_list: D004272:DNA, Mitochondrial; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific