Mitochondria are the tiny cellular “power plants” in our cells, which take energy from our food and convert it into a form that can be used to power the cell’s energy-intensive processes. Like other power plants, they generate waste in the process – in this case, free radicals – which over time damage mitochondrial DNA. As a result, a small but rising number of our cells get taken over by such dysfunctional mitochondria as we age. These damaged cells in turn export toxic molecules to far-flung tissues, contributing to Parkinson’s disease, age-related muscle dysfunction, and other conditions.
The MitoSENS goal is to achieve a grand engineering solution to the problem of accumulation of cells with these mutation-bearing mitochondria: allotopic expression of functional mitochondrial genes. Allotopic expression involves placing “backup copies” of all of the protein-coding genes of the mitochondria in the cell’s nucleus. From this “safe harbor”, the copied genes can then direct the cell’s machinery to produce engineered versions of the missing mitochondrial proteins and deliver them to the mitochondria. With their full complement of proteins restored, mitochondria can resume producing energy normally, despite lacking the genes to produce them on their own.
In 2016, the MitoSENS team achieved a major breakthrough in successfully demonstrating efficient replacement of the missing mitochondrial ATP8 gene in cells from a human patient with an ATP8 mutation, restoring their ability to produce energy using the most efficient pathway.
After significant work to extend 2016’s breakthrough to other genes, the team discovered that an established method already widely used in biotechnology could also be applied to enable significantly more consistent production of allotopically-expressed protein.
To test this novel method more broadly, the MitoSENS group first briefly allotopically expressed each of the thirteen vulnerable mitochondrial genes via a transient loop of DNA located in the cytosol. Versions of the genes engineered the new way produced a great deal more RNA (the “working copies” of the gene that the cellular machinery uses to make protein) than the same genes engineered in the way that all previous investigators have used.
All thirteen of the genes engineered in this new way were able to produce actual protein, versus only a fraction of the conventionally-engineered genes. This milestone achievement is being prepared for publication in a scientific journal as of this writing, and tests are now underway to verify that all proteins thus expressed are properly incorporated into the mitochondria’s energy-production system.
The team has compared performance between ‘traditional’ and novel systems for producing allotopic ATP8 in cells derived from FVB mice. These mice bear a minor but significant mutation in ATP8 that causes functional problems, e.g., a tendency to poorly metabolize incoming blood sugar after a meal. The cells engineered using this novel method produced significantly more ATP8 protein than those engineered the conventional way – and it is important to note that in this experiment, the new genes were actually cemented into the nucleus and expressed from there, thus mimicking the goal for human MitoSENS therapies. The allotopically-expressed protein works as intended when using the improved system: it enters the mitochondria, incorporates properly into the energy-producing machinery, and significantly enhances these cells’ ability to survive when they are forced to rely on the mitochondria’s primary energy-generation mechanism.
Next, the MitoSENS team plans to demonstrate efficacy in living, breathing mice – specifically, Maximally Modifiable Mice (MMM) from the SRF funded work at ASC. The new MMM-derived mouse model will have the allotopic ATP8 construct engineered into their nuclear genomes from conception, but will have mitochondria (and thus mitochondrial DNA) derived from FVB mice, with their mutant ATP8 gene. This work, in conjunction with behavioral studies to be performed in collaboration with the Brand lab at the Buck Institute, is expected to prove that the allotopic gene actually functions in vivo, restoring the mice’s ability to generate cellular energy efficiently.