Fight Aging!

Mitochondria, hundreds to a cell, are evolved descendants of the ancient bacteria that became symbiotic with the first, primitive cells. Mitochondria still behave a great deal like bacteria, in that they fuse together, replicate, carry a small circular genome, the mitochondrial DNA and promiscuously swap component parts. While the primary role of mitochondria is the generation of adenosine triphosphate (ATP), a chemical energy store used to power cell processes, they are also well integrated cellular components in a broader sense, influential in a range of fundamental cellular activities.

Mitochondrial DNA is a lot smaller than it once was. Evolution has gradually shifted mitochondrial genes into the nuclear DNA resident in the cell nucleus, all except a handful of remaining genes. Arguably the thirteen genes that remain are those for which there is no easy path for evolutionary mechanisms to produce a move into the nucleus. Yet we would like to move those genes into the nucleus, as damage to mitochondrial DNA is likely a major contribution to degenerative aging. Mitochondrial DNA is less well protected and repaired than nuclear DNA, and mutations can lead to mitochondria that are both dysfunctional and able to outcompete their undamaged siblings. The cell suffers as a result. Having a backup copy of a mutated gene that can supply proteins from the nucleus would evade the consequences of damaged mitochondrial DNA.

Finding ways to move the remaining mitochondrial genes into the nucleus, a process known as allotopic expression, is a long-running initiative at the SENS Research Foundation. After years of work, the research team there achieved success with only one of the genes so far, ATP8, and we can add another equally hard-won success by Gensight Biologics for ND4. That leaves another eleven genes that so far have proven resistant. The challenge is not inserting copies into the nucleus, as that is easy to accomplish in a research setting. The challenge lies in finding the alterations that will allow functional, correctly folded proteins to make their way back to the mitochondria where they are needed. As noted, this is something that evolution has failed to achieve, for reasons that we might guess at, but remain unclear.

The latest crowdfunded project to be proposed by the SENS Research Foundation is to use screening of hundreds of thousands of genetic variants of the COX2 mitochondrial gene to seek insight into approaches that will work for the allotopic expression of this and other mitochondrial genes. I think that this is a worthy endeavor, and I donated a modest amount to the project. Screening of very large numbers of options is a good way forward where years of more rational step by step design have failed to cover enough of the possible paths to find success, especially when it can be accomplished for a reasonable cost. The laboratory tools of genetics and gene therapy cost little these days.

Finding a Cure for Mitochondrial DNA Diseases through COX2 Variations to Restore Cell Function

Mitochondrial DNA is highly prone to mutation due to a variety of factors and these mutations result in several pathologies. Our endeavor is in identifying gene therapy approaches to address these mutations. The COX2 gene is a core component of Complex IV in the oxidative phosphorylation relay. Mutations in this gene are associated with Complex IV deficiency affecting a critical step in the oxidation of cytochrome C using molecular oxygen.

Our lab has successfully produced proteins for all 13 genes found in mitochondria by allotopic expression, but only one (ATP8) has successfully restored function in a disease-model cell line. Natural evolution has already transferred more than 1000 genes from mitochondrial DNA to the nucleus. Our goal is to find variants of the COX2 gene that can help cells function properly by mimicking the natural evolutionary process. Results from such an experiment would not only be a significant step towards efficacious COX2 gene therapy but would also provide key insights into the genetic changes necessary for successful allotopic expression of all mitochondrial genes.

We propose to generate more than 1 million variants for the COX2 gene using error-prone PCR similar to a study performed in yeasts and test these variants in rescuing oxidative phosphorylation, a function that is crucial for ATP production in cells. The nutrients that we consume are metabolized to CO2 and high-energy electron donors that further combine with oxygen to convert ADP to ATP. We will test this in a human model cell line that is lacking the COX2 protein. This cell line is unable to grow in nutrient medium restrictive for oxidative phosphorylation. Upon placing the variants in such a medium, only cells that are capable of performing oxidative phosphorylation can survive. Such a screen would allow us to identify functional variants of the COX2 gene for further analysis.