Mitochondrial diseases are commonly caused by mutations in the mitochondrial DNA (mtDNA), which in most cases co–exists with the wild–type (mtDNA heteroplasmy). We have engineered TAL–effector nucleases (TALENs) to localize to mitochondria and cleave different classes of pathogenic mtDNA mutations. MitoTALEN expression led to permanent reductions in deletion or point mutant mtDNA in patient–derived cells, raising the possibility that they can be curative to some of these diseases.

SUMMARY Mitochondrial diseases include a group of maternally inherited genetic disorders caused by mutations in mitochondrial DNA (mtDNA). In most of these patients, mutated mtDNA coexists with wild type mtDNA, a situation known as mtDNA heteroplasmy. Here we report on a strategy towards preventing germline transmission of mitochondrial diseases by inducing mtDNA heteroplasmy shift through the selective elimination of mutated mtDNA. As a proof of concept we took advantage of NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB, and selectively prevented their germline transmission using either mitochondria targeted restriction endonucleases or TALENs. In addition, we successfully reduced human mutated mtDNA levels responsible for Leber’s hereditary optic neuropathy (LHOND), and neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP), in mammalian oocytes using mito-TALENs. Altogether, our approaches represent a potential therapeutic avenue for preventing the transgenerational transmission of human mitochondrial diseases caused by mutations in mtDNA.

Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules. Using a mouse model with a heteroplasmic mtDNA mutation, we tested whether mitochondrial-targeted TALENs (mitoTALENs) could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNA levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice.

Cytochrome c oxidase contains two redox-active copper centers (Cu A and Cu B ) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the Cu A center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1⅐COX4⅐COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1⅐COX4⅐COX5A subassembly, suggesting that MTCO2 associates with the MTCO1⅐COX4⅐COX5A subassembly after the Cu A center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1⅐COX4⅐COX5A subassembly.