DNA-editing enzymes as potential treatments for heteroplasmic mtDNA diseases.

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild-type and mutant mtDNA molecules co-exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA-editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.

Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease.

Mitochondrial diseases are frequently caused by heteroplasmic mitochondrial DNA (mtDNA) mutations. As these mutations express themselves only at high relative ratios, any approach able to manipulate mtDNA heteroplasmy can potentially be curative. In this study, we developed a system to manipulate mtDNA heteroplasmy in all skeletal muscles from neonate mice. We selected muscle because it is one of the most clinically affected tissues in mitochondrial disorders. A mitochondria-targeted restriction endonuclease (mito-ApaLI) expressed from AAV9 particles was delivered either by intraperitoneal or intravenous injection in neonate mice harboring two mtDNA haplotypes, only one of which was susceptible to ApaLI digestion. A single injection was able to elicit a predictable and marked change in mtDNA heteroplasmy in all striated muscles analyzed, including heart. No health problems or reduction in mtDNA levels were observed in treated mice, suggesting that this approach could have clinical applications for mitochondrial myopathies.

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

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 (DSBs) 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 this 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 show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a 'differential multiple cleavage-site' model.

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a 'differential multiple cleavage site' model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.

Histoenzymology of oxidases and dehydrogenases in peripheral blood lymphocytes and monocytes for the study of mitochondrial oxidative phosphorylation.

Histoenzymological methods usually performed on muscle fibres have been adapted to assess the functioning of oxidative phosphorylation in human circulating blood lymphocytes and monocytes. Oxidases and dehydrogenases were analysed in lymphocyte/monocyte smears. The specificity of each histoenzymological reaction was tested using a specific respiratory chain inhibitor: rotenone for NADH diaphorase, thenoyltrifluoroacetone for succinate dehydrogenase, potassium cyanide for cytochrome c oxidase and oligomycin for ATPase. Complex I activity was detected, but inhibition with rotenone was incomplete. Complexes II, IV and V were almost completely inhibited. These observations indicate that histoenzymology is a valuable method for detecting the activity of these oxidative phosphorylation enzymes in lymphocytes and monocytes. The histoenzymology tests performed on fresh peripheral blood cells resembled those used for muscle biopsies. They could be useful for the diagnosis of respiratory chain disorders in patients.

Human primary Sjögren's syndrome autoantibodies as mediators of nitric oxide release coupled to lacrimal gland muscarinic acetylcholine receptors.

IgG obtained from sera of primary Sjögren's syndrome (pSS-IgG) patients and its interaction with M3 muscarinic cholinoceptors of rat exorbital lacrimal glands were studied by indirect immunofluorescence (IFI) and binding assay. Primary Sjögren's syndrome IgG stained epithelial cells with a continuous fluorescence pattern. The IFI imagen was attenuated by incubating the pSS-IgG with a synthetic peptide corresponding to the second extracellular loop of M3 muscarinic cholinoceptor. Primary SS-IgG was also able to bound irreversibly to muscarinic acetylcholine receptors (mAChRs) displacing the specific cholinergic antagonist QNB. Moreover, these antibodies triggered intracellular signals coupled to M3 muscaric cholinoceptors such as nitric oxide synthase (NOS) activation and cGMP production. Both primary Sjögren's syndrome IgG effects mimicked carbachol action and were abrogated by specific muscarinic antagonist 4-DAMP. The nitric oxide pathway through muscarinic cholinoceptors activation by pSS-IgG on rat exorbital lacrimal gland is also described. We proposed that chronic interaction of these autoantibodies on lacrimal gland muscarinic acetylcholine receptors could lead to tissue damage through nitric oxide release after immunological stimulation.