Mitochondria, the power plants of the cell, are the evolved descendants of symbiotic bacteria. They still carry a remnant of the original bacterial DNA, encoding a few vital genes. That mitochondrial DNA becomes damaged in aging, and based on the various direct and indirect evidence for the size of the influence of mitochondria on life span, this mutational damage and its consequences are a big deal. Some way to revert mitochondrial DNA damage is high on the list of rejuvenation therapies that we would like to see developed in the years ahead.
The open access paper here is largely focused on the treatment of inherited mitochondrial conditions, in which a sizable fraction of mitochondrial DNA in every cell throughout the body is affected by the same specific harmful mutation. It is nonetheless is a useful tour of some of the available tools that researchers might consider adapting in order to attempt to reverse the mitochondrial damage that occurs in aging. It mentions allotopic expression (copying mitochondrial DNA into the cell nucleus as a backup) only briefly, but that is fine: the audience here is no doubt familiar with this favored strategy of the SENS research program, but possibly less familiar with the other options on the table that involve mitochondrial DNA.
Most of those options boil down to either (a) delivering or creating larger amounts of correct mitochondrial DNA or (b) destroying as much broken mitochondrial DNA as possible. Both are viable approaches for inherited mitochondrial disease, but the dynamics are different in the case of aging. The real challenge posed by the most harmful age-related mitochondrial DNA mutations is that they result in mitochondria that are both broken and able to replicate more effectively than their peers. So even a single copy can quickly replicate to take over a cell, and that places tough constraints on the ability to produce benefits through treatments that work through the methods noted above. Adding correct mitochondrial DNA seems non-viable in principle on its own, while methods of destroying broken mitochondrial DNA would have to be exceedingly efficient to make any lasting progress. Still, the latter may be worth testing in order to certain, given that the technology exists to make the attempt.
The human mitochondrial DNA (mtDNA) is a small double stranded circular genome which is maternally inherited. Each mammalian cell contains in average one thousand copies of mtDNA and each molecule contains 37 genes. Defects in the mtDNA, both point mutations and large scale rearrangements, have been associated with severe mitochondrial syndromes. When pathogenic mutations occur in the mtDNA most often both mutant and wild-type copies co-exist within the same cell, a phenomenon known as heteroplasmy, and, in general, only when the mutation load is higher than approximately 80% symptoms manifest.
Currently, there are no effective strategies to cure mitochondrial disease and, in spite of the advances in genetics and biotechnology, there are still some gaps in the understanding of mitochondrial genetics. For instance, we do not know what controls mtDNA copy number, and mechanisms of mtDNA replication are still controversial. During the past 16 years our lab and others have been focusing in the use of endonucleases to target mitochondria and induce double strand breaks (DSB) in the mtDNA. Taking advantage of the fact that mitochondria lack an established DSB repair mechanism, it has been shown that mtDNA is quickly degraded after a DSB. Therefore, heteroplasmy can be manipulated and the mutant genomes can be efficiently eliminated through cleavage of mutant mtDNA and repopulation of the cells with wild-type mtDNA. Recently, a new door has been open regarding translation of these techniques into the clinics by the development of precise DNA editing tools, which can be targeted to mitochondria to promote DSB in the mtDNA.
Because of the high rate of mutations in the mtDNA, new pathogenic mutations are recurrently introduced into the human population. Recently, next-generation sequencing technology has been used to identify and quantify mtDNA mutations. However, these techniques have a high intrinsic error rate when applied to detection of low-level heteroplasmy. Despite all the technological difficulties, it is believed that mtDNA heteroplasmy exists in almost every healthy individual studied, even though at very low levels. These heteroplasmic variants can also be passed down the maternal lineage, raising the possibility that some presumably somatic mutations measured late in life are actually low-level heteroplasmies that have been inherited and somehow clonally expanded.
In contrast to point mutations, primary mitochondrial rearrangements of mtDNA are not inheritable, they are sporadic. Large-scale deletions are typically heteroplasmic and result in disease. To date, roughly 120 different mtDNA deletions have been found in patients with mitochondrial disease. In this case, the heteroplasmic threshold is reported to be lower than the one for point mutations, the patients manifest the disease symptoms with as low as 50-60% heteroplasmic mtDNA levels. Two different models arise to explain deletions in the mtDNA, while one points to replication errors, the other one points to poor and inefficient mtDNA repair mechanisms.
The concept of shifting the balance between healthy and mutated mtDNA as a treatment for heteroplasmic mtDNA disease has been under investigation over the past 20 years. Many publications demonstrated that it is possible to manipulate the mtDNA and shift heteroplasmy, either in vitro or in vivo. By simply reducing the levels of the mutant allele below a certain threshold, an improvement in pathology is achieved. There are currently at least two strategies for applying gene therapy to patients with mtDNA diseases: 1) Allotopic expression of mitochondrial genes; 2) Manipulation of mtDNA heteroplasmy. Allotopic expression of mitochondrial genes which consists in the synthesis of a wild-type version of the mutated protein in the nuclear-cytosolic compartment followed by its import into mitochondria has been a controversial approach because of the high hydrophobicity of mtDNA-encoded proteins and the competition with endogenous counterparts. Nonetheless, clinical trials for Leber's optic neuropathy are currently ongoing. The use of mitochondrial endonucleases is still in its infancy, but hopefully will move into the clinics in the next few years.
To conclude, the manipulation of mtDNA heteroplasmy either by using mito restriction endonucleases, mito zinc-fingers or mitoTALENs could facilitate delivery and increase specificity of mtDNA editing, having the potential to eliminate mutant mitochondrial genomes from germline treated affected patients.