Free radicals derived from our cells’ energy-producing mitochondria can mutate the organelle’s DNA, leading to deletions of large stretches of the mitochondrial genome. These deletion mutations prevent the mitochondria from building various pieces of the electron transport chain (ETC), by which mitochondria generate most cellular energy. The accumulation of deletion-mutation-containing cells is a significant consequence of aging, and is implicated in age-related disease as well as in several currently incurable inherited mitochondrial disorders.
The SRF-RC mitochondrial mutations team is moving forward on a method for targeting engineered nuclear-encoded genes (that could function as “backup copies” for cells with deletion mutations) to the mitochondria, and for furthermore optimizing the precision of this targeting. The “working copy” of the relocated mitochondrial gene in this method is equipped with two special sequences. One “untranslated” sequence is not turned into a protein itself, but helps protect the engineered protein during the import process. The other, called the mitochondrial targeting sequence, is a tag appended to the final protein following expression that allows it to be imported once expressed. Combining the two sequences allows the “backup copies” of genes to be turned into working copies in the cell nucleus; to have the “working copies” targeted to the surface of the mitochondria to be decoded and turned into protein. Even as it is still in the process of being decoded, the emerging protein is quickly directed to the surface of the mitochondria for import and incorporation into the ETC, restoring mitochondrial function.
In 2013, the SRF-RC mitochondrial mutations group created two new cell lines which are 100% null for two mitochondrially-encoded genes: ATP8 and CYB. Using these two new cell lines, this year the SRF-RC mitochondrial mutations team was finally able to unleash their engineered ATP8 gene in cells whose mitochondria completely lack the ability to generate the corresponding proteins on their own. The team expects to be able to announce a dramatic rescue of such “ATP8 null” cells using their protein targeting strategy very soon. They anticipate that these results will deliver the proof-of-concept for the overall approach, which should then be applicable as a rescue platform for all thirteen mitochondrially-encoded proteins.
Further work by the team aims to enable delivery of working instructions for building proteins that can keep the ETC intact and functioning in the event of age-related mutations of the original mitochondrial genes for these proteins. This method utilizes a “borrowed” structure already employed by mitochondria to take in RNA from the main body of the cell. The team has now achieved the critical first benchmark — i.e. delivering any RNA into the mitochondria — in this pioneering work using a convenient (but not naturally mitochondrially-expressed) RNA.
Mitochondria: The “Powerhouse” of the Cell
Mitochondria provide energy for the cell by synthesizing energy in the form of high energy bonds. This energy synthesis occurs through a process called oxidative phosphorylation in which respiratory enzymes in mitochondria convert a molecule called adenosine diphosphate (ADP) into the energy currency of the cell, ATP. One interesting feature of mitochondria is that they contain their own DNA (mtDNA). As cells and mitochondria have co-evolved, most of this genetic information has been transferred to the nucleus, leaving only thirteen protein-encoding genes in the mtDNA.
The Problem: Energy Production Leads to mtDNA Mutations Accumulating Over Time
Housing these thirteen genes within the mitochondria themselves is precarious because the conditions required to synthesize ATP create reactive oxygen species. Over time, these toxic free-radical byproducts damage the mitochondrial genes in more and more cells, compromising respiratory chain function and hence energy production. The accumulation of mutations in mitochondrial DNA is implicated in the metabolic derangement of aging and in accelerating the course of the degenerative aging process as a whole. One need only examine clinical manifestations of mitochondrial genetic diseases to see the similarities they share with the maladies of aging. For example, mutations in the gene ND1 have been implicated in the development of Parkinson’s disease, and Cytochrome B (CYB) mutations can cause muscle fatigue/ exercise intolerance in young patients.
SRF’s strategic approach to this problem is to engineer a way to let mitochondria keep producing energy normally, even after mitochondrial mutations have occurred. Although damage to mitochondrial DNA is inevitable so long as it is housed in the mitochondria, the harmful effects of mitochondrial mutations can be bypassed by engineering backup copies of the thirteen protein-encoding genes and housing the copies instead in the nucleus of the cell. These allotopic gene copies could continue to provide the necessary proteins even when mutations have compromised the mtDNA’s ability to do so. Moreover, the nuclear gene copies would be better shielded from damaging toxins and better maintained by DNA repair machinery. Since the majority of mitochondrial proteins are naturally nuclear-encoded, the natural mechanism to deliver the allotopically-expressed genes to the mitochondria can be co-opted.
Overcoming Obstacles to the Allotopic Expression Strategy
Previous attempts at allotopic expression have experienced only limited success due to problems with import of the proteins into the mitochondria, which were likely caused by the hydrophobic nature of the proteins. In addition to being ineffective, incomplete protein import is likely toxic to the mitochondrion and ultimately to the cell. To overcome these problems and achieve optimal mitochondrial import, SRF plans to use a co-translational import strategy. This strategy has been successfully employed by Professor Corral-Debrinski in work previously funded by SENS Research Foundation. The improvement that Dr. Corral-Debrinski has pioneered is tagging the RNA of the genes with sequences that not only target the proteins to the mitochondria but also direct the RNA to the mitochondrial surface before it is translated into protein. This approach prevents the convoluted folding of these proteins during translation in the watery environment of the cytosol, improving the efficiency of protein import.
Therapeutic Significance of the MitoSENS Project
Using mitochondrial gene therapy as a strategy to correct mitochondrial dysfunction has many advantages. In theory, such gene therapy could be used both to prevent and to correct the effects of mitochondrial mutations. Another exciting potential is that any therapy we develop could, in principle, be used to treat any of the known diseases of the mitochondria, such as LHON (Leber Hereditary Optic Neuropathy), Leigh syndrome, and NARP (Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa), all of which are debilitating diseases. Indeed, allotopic expression is already being tested in human clinical trials to treat LHON. Treatment of these known and well-characterized diseases is a licensable therapeutic indication under current regulation, which therefore constitutes an entry point for the first human uses of mitochondrial gene therapies that emerge from our work.