We recently had the opportunity to interview Dr. Amutha Boominathan from the SENS Research Foundation, at the Ending Age-Related Diseases 2019 conference about her research on mitochondrial repair therapies, the value of animal models, and her views on the future of aging research.

Dr. Amutha Boominathan received both her MSc and her PhD in Biochemistry from the University of Pune and the National Chemical Laboratory in India, respectively. She went on to do postdoctoral work in the U.S. relating to mitochondrial biogenesis at U. Penn and Rutgers University. She has extensively studied mechanisms of fusion and fission in mitochondria, Fe-S cluster biosynthesis, and protein import into mitochondria as part of her postdoctoral fellowship with the American Heart Association.

Currently, Amutha leads the MitoSENS program at SENS Research Foundation in Mountain view, California. Her research group is focusing on understanding mitochondrial DNA (mtDNA) mutations and restoring lost functionality as a result of these mutations by way of the allotopic expression of mitochondrial genes. Inherited mtDNA mutations can result in severe and debilitating diseases, such as NARP, Leigh’s syndrome and MELAS. Even in otherwise healthy individuals, mtDNA mutations accumulate with age. The MitoSENS team has already succeeded in stably expressing the ATP8 gene using their method and is looking forward to tackling each of the 13 mitochondrial protein genes in the coming years. Its goal is to develop safe and effective gene therapies for mitochondrial dysfunction.

Your research group started developing an improved method for allotopic expression of mtDNA in 2015 that has already shown very promising results. What hurdles for allotopic expression does this new method overcome and what do you think that means for further studies in animal models?

The major hurdle that we have overcome is, at least, showing protein products for all the 13 genes. We made some fundamental changes to all 13 genes with a uniform approach, but that approach may not work (equally well) for all of them. We may have to engineer each one of them for specific properties.







So, all of these 13 genes differ with respect to their length, their hydrophobicity, and the complexes that they target. The main hurdle is actually the hydrophobicity factor. Protein products span the mitochondrial inner membrane multiple times, and you are actually targeting these proteins from the “opposite” side. See, these 13 proteins are (normally) synthesized within the matrix, and they are inserted into their complexes. But, in allotopic expression, they are synthesized in the cytosol and have to traverse two membranes and then go to the right location. Now, a mitochondrion by itself has translocases of the outer membrane and translocases of the inner membrane, and it has multiple pathways. Depending on the location where these proteins have to go, there are different mechanisms in play [1].

We will have to engineer them one after the other or modify them in such a way that it recognizes the right pathway. So, like I said, we are causing global changes to all 13 genes, and we will cause specific changes to each one of them to make it functional (as a whole). The first step is to at least see a product, and that’s what we’ve overcome now.

What have been MitoSens’ criteria for selecting mtDNA genes to work on for allotopic expression?

One of the other hurdles is proving that your technology actually works, and for that, you need model systems. The reason we were able to show that ATP8 really works is because we were able to get a patient cell line with a severe mutation that’s null for the ATP8 protein. Usually, in humans, mutations (to mitochondrial genes) manifest in various levels, but it is unusual that the protein is completely absent in the patient. It’s a rare event. But mitochondrial DNA exists in heteroplasmy. There are wild type and mutant levels, both present continuously, and it’s the tipping factor that causes a disease phenotype to ensue. Or it keeps itself in control, where the wild-type mtDNA overpowers the mutant DNA.

The one reason we were able to really convincingly show ATP8 works is because we were able to get the null cell line and show that the exogenous protein goes into the right location and regains many of the functions that were absent before. Basically, you have the cell line available, which is really rare. So, let’s make use of it.







A review published in April this year by a group of Chinese researchers [3] discussed the specific benefits of using Drosophila flies as a model for research on mtDNA mutations. Can you explain why the MitoSENS group is choosing to perform their upcoming research with mice rather than flies?

Like we heard at this conference, flies, at the biochemical level, may be able to show that certain things work, but you need higher mammalian models or animal models before you can take any type of intervention to the human clinic. Again, it so happens that there is a mouse disease model available for the ATP8 gene. This is a very good model in the sense that it doesn’t have a null mutation; the protein is still there, but it is a lower-functioning protein.

The phenotypes are subtle but very important. They are diabetic or insulin resistant. Behaviorally, they stress out very easily. So, if the allotopic ATP8 really works, and if we are able to express it from the nucleus in this mouse and recapitulate some of the functions there, it’ll be easy to show that it comes back, both from a behavioral and biochemical point of view. That’s why we prefer the mouse model.

Why does developing and using the Maximally Modifiable Mouse in aging research represent a significant step toward catalyzing the delivery of rejuvenation biotechnologies?

As you know, SENS actually funded the Maximally-Modifiable Mouse. Gene therapy, for now, is normally done using AAV vectors, which is still a transient system. Even today, we saw Dr. Blasco talk about it. There are advantages to it being transient: it kind of gets diluted with time. But, in an allotopic context, we want to express them stably and continuously. Now, what the Maximally-Modifiable Mouse allows us to do is put in a large amount of DNA. In the AAV scenario, you are limited by the size of the payload that you can put in the vector. Our goal is to eventually place all the 13 genes in one place. Yeah, it is a high goal, but that’s what we want to achieve.







To work towards that, we have made this Maximally-Modifiable Mouse so we can put our one gene in there now, and as we work on other genes, we want to (eventually) integrate all of them in one place, where we can control their transcription and translation as our other, nuclear mitochondrial genes are controlled. Ultimately, our goal is to achieve that kind of regulation. We’re better able to do that with the Maximally-Modifiable Mouse model.

An alternative to allotopic expression is the xenotropic expression of proteins from other species functioning in similar pathways. An example of successful xenotropic expression has been seen in sea squirt alternative oxidase, which can completely rescue the viability of certain Drosophila mutants [4]. Would you say that allotopic expression has marked benefits over xenotropic expression when thinking about translation of therapies to humans?

What we want is to keep it as humanized as possible. So, these (genes) are, after all, foreign for the nuclear genome; you may already be introducing new immune profiles that this foreign gene will generate. Now, if you want to do a xenotropic expression, that’s going to introduce even more changes. From a testing point of view, we do check all the other genes from other organisms that have already migrated to the nucleus. We may make similar changes, but we want to keep it as humanized as possible.

In migrating to the nucleus over the course of evolution, a lot of these genes have acquired different changes that permit them to be transformed. In certain animals, Complex I actually functions by way of just one protein, for example NDI1 in yeats. But, in the human context, it needs 47 proteins, seven of which come from the mitochondrial DNA, and over 40 of them come from the nuclear DNA [6].

You don’t want to express this one protein (NDI1) and then try to regain function. From a purely experimental point of view, you can do it, but if you’re going to put it in humans then that’s not the best way to go. You want to preserve the integrity of the complex. I also can’t imagine what kind of regulatory hurdles that would introduce. Gene therapy itself is difficult enough, but imagine gene therapy with something like a yeast gene.







What do you think will be a realistic timeframe for therapies targeting mtDNA mutations to reach humans?

They are actually already doing that but with the recoded version. That means we already have a precedent. All we have to show is that our version of it is better and that ours has a better immune profile. That’s also why we want to do it in animal models, so we can actually show how it’s better. I don’t know about the time-frame; that’s a very difficult question. During the conference, somebody asked me (about it). If the animal studies go well, I want to say five years. Not five years before it reaches people, but five years to establish enough proof of principle that we can start to develop this for people.

There are several factors that predispose mtDNA to accumulate mutations over time. Would it be necessary to supplement allotopic expression of mtDNA genes with other types of therapies to decrease the number of mutations built up over time in order to see significant effects on aging?

It is a good question. If you supplement allotopic expression with what is available now, like Idebenone, Elamipretide, whatever, it will be beneficial. They are all antioxidants, and they improve the health of the OxPhos function.

However, if you move to gene therapy directly in patients, their disease state may not be conducive to accept that therapy. Their complexes are not completely functional, but that’s part of a cascade, so, really, the entire mitochondrial function is not the way it’s supposed to be. You might want to improve their disease state to a certain level, even if it is subpar, and then apply the gene therapy, because you want to ensure that the gene therapy is useful to them.







With regard to aging, it’s a little controversial whether mtDNA mutations are part of the root cause of aging or not. Our goal here is to help patients first, and if it works, we can extend from there.

In your view, what does aging research need most right now to ensure it can make the most significant leaps that the field is capable of in the coming 10 years?

I think you need good biomarkers. That’s lacking in the field. Everybody wants to have a quick fix. They have all these different areas that they think are very important to aging, but I don’t think that’s the way it is. I think it’s more like a general breakdown of everything with time. So you need better markers, and maybe even a better mindset where it’s okay to be healthy (in old age). People shouldn’t be resigned to the fact that they will age with time and that they are going to die. Maybe a little more public education is needed to accept that it’s okay to want and to have a healthy lifespan.

Is there a question that no journalists ever ask you that you would like us to ask you?

That’s a surprisingly difficult question. I don’t actually have an answer for that right now. I do want to say something about the MitoMouse campaign, though. From the MitoTeam at SENS, I want to express my gratitude to LEAF for helping us with that.







We would like to thank Dr. Boominathan for taking the time to interview with us. The MitoSENS team led by Dr. Boominathan has also just launched MitoMouse, a follow-up to its 2015 MitoSENS project on our research crowdfunding platform Lifespan.io. The team is aiming to create a proof of concept for mitochondrial repair therapies, and your contributions are critical in helping to bring this vital and potentially life-saving research to the clinic more quickly.

References







Pfanner, N., Warscheid, B., Wiedemann N. (2019). Mitochondrial proteins: from biogenesis to functional networks. Nature Reviews: Mol Cell Bio, 20, 267-284.

Stefano, G.B., Bjenning, C., Wang, F., Wang, N., Kream, R.M. (2017). Mitochondrial Heteroplasmy. Advances in experimental medicine and biology, 982, 577-594.

Chen, Z., Zhang, F., Xu, H. (2019). Human mitochondrial DNA diseases and Drosophila models. Journal of Genetics and Genomics, 46(4), 201-212.

Chen, Z., Qi, Y., French, S., Zhang, G., Covian Garcia, R., Balaban, R., Xu, H. (2015). Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila reveals novel aspects of mitochondrial regulation and function. Mol. Biol. Cell, 26, 674-684.

Boominathan, A., Vanhoozer, S., Basisty, N., Powers, K., Crampton, A.L., Wang, X., Friedricks, N., Schilling, B., Brand, M.D., O’Connor, M.S. (2016). Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant. Nucleic Acids Research, 44(19), 9342–9357.







Brandt, U. (2006). Energy converting NADH:quinone oxidoreductase (complex I). Annual Review of Biochemistry, 75, 69–92.