We interviewed Dr. Amusa Buminasan of the SENS Research Foundation at the Ending Age-Related Diseases 2019 conference about her research on mitochondrial restorative therapy, the significance of model animal experiments, and her views on the future of aging research.
Dr. Amusa Buminasan received her Masters and PhD in Biochemistry from the University of Pune and National Chemical Laboratory in India, respectively. She continued to research mitochondria at the University of Pennsylvania and Rutgers University in the United States. She studied in detail the mechanisms of fusion and division in mitochondria, the biosynthesis of iron-sulfur clusters and the delivery of proteins to mitochondria as part of her post-dock internship at the American Heart Association.Amusa currently leads the MitoSENS program in Mountain View, California. Her research team is studying mitochondrial DNA (mtDNA) mutations and developing methods to restore the functionality lost as a result of these mutations by allotopic expression of mitochondrial genes. Hereditary mtDNA mutations can lead to severe and debilitating diseases, such as NARP, Lei syndrome, and MELAS.Using its technologies, the MitoSENS team has already succeeded in stable expression of the ATP8 gene, and is looking forward to repeating success for all 13 genes in the near future. Their goal is to develop safe and effective gene therapy for the treatment of mitochondrial dysfunction.Interview
Thomas Warner Lehner : Your research team began developing an advanced method for the allotopic expression of mtDNA in 2015, and it has already shown very promising results. What obstacles to allotopic expression does this new method overcome and what, in your opinion, does this mean for further research on model animals?Amusa Buminasan : The main obstacle that was overcome was that we at least expressed all 13 genes. We used a single approach to make some serious corrections to all 13 genes, but this approach may work differently for them. Perhaps we will have to redesign the indicated approach for each gene because of their features.So, all 13 genes differ in length, hydrophobicity, and the complexes they target. The main obstacle is the hydrophobicity factor. Proteins cover the inner mitochondrial membrane in several layers, and you need to get them from the outside. These 13 proteins are synthesized in a matrix and integrate into their complexes. But with allotopic expression, they are synthesized in the cytosol and must pass through two membranes, and then get to the right place. Mitochondria itself has translocases (enzymes that catalyze the transfer of ions or molecules through membranes) of the outer and inner membranes, which work differently. Depending on the destination, different mechanisms will be involved.We must develop the proteins sequentially, one after the other, or change each of them so that they recognize and engage the correct path. We will make general corrections to all 13 genes, and in addition, we will make personal corrections to each of them to make them functional. The first step is to at least see the result - and this step has been completed.Thomas Warner Lehner : What criteria guided MitoSENS when choosing genes to work on allotopic expression?Amusa Buminasan: One of the obstacles is checking that our technology really works, and for this we need model systems. The reason that allowed us to show that ATP8 works is the presence of a patient cell line with a serious mutation that is zero for ATP8. MtDNA mutations in humans appear at different levels, but it is rather unusual for a patient to completely lack some protein. This is a rare event. But mitochondrial DNA exists in heteroplasmy . The ratio of mutant and normal mitochondria is a factor determining the phenotype of the disease. Or the disease is restrained due to the fact that normal mtDNAs suppress mutant DNA.One of the reasons that we were convinced of the correct functioning of ATP8 is that we managed to get a zero cell line and show that the exogenous protein gets to the right place and restores many of its functions. In fact, the right cell line is available to us - a rare success. Well, why not take advantage of this?Thomas Warner Lehner : A review published in April this year by a group of Chinese scientists discussed the benefits of using Drosophila flies as a model for studying mtDNA mutations. Can you explain why the MitoSENS group prefers to conduct its research on mice rather than flies?Amusa Buminasan: As we learned at this conference, flies, at the biochemical level, make sure certain methods work, but you need higher mammals to go on to clinical trials in humans. Again, it so happened that we have a model of mouse disease for the ATP8 gene. This is a very good model without a zero mutation; the protein is still available, but it is a low-functional protein.Symptoms of the disease are mild, but very important. They are characteristic for diabetes or insulin resistance. From a behavioral point of view, mice are not stress resistant. Due to this, if allotopic ATP8 works and if we can synthesize it in the mouse’s cell nucleus and restore its functions, then we can easily demonstrate that the state of the body has returned to normal, both from a biochemical and behavioral point of view. That is why we prefer the mouse as a model animal.Thomas Warner Lehner : Why is the development and use of the Maximally Modifiable Mouse a significant step in the development of anti-aging therapy?Amusa Buminasan: As you know, SENS is funding the Maximally Modifiable Mouse. Currently, gene therapy is usually carried out using vectors of adeno-associated viruses, their therapy is temporary. Even now, Dr. Blasco has confirmed this. There are advantages in that the effect is temporary - it seems to weaken in time. But in the context of allotopic expression, we would like the effect to be stable and permanent. Thanks to the Maximally Modifiable Mouse, we can put whole genes into the genome. Using adeno-associated viruses, you are limited by the payload that you can contribute to the genome. And our goal is to introduce all 13 genes into the genome over time. This is a high bar, but that is precisely what we want to achieve.To achieve this, we created the Maximally Modifiable Mouse in order to place at least one gene in it. We want, in the future, to place other genes in its genome so that their transcription and translation are controlled in the same way as other mitochondrial genes located in the nucleus are controlled. The Maximally Modifiable Mouse is best suited for this.Thomas Warner Lehner: An alternative to allotopic expression is xenotropic expression of proteins of other species that function in a similar way. An example of successful xenotropic expression has been shown in alternative ascidia oxidase, which completely restored the viability of some mutated Drosophila. Could you say what are the advantages of allotopic expression compared with xenotropic expression when it comes to the use of therapy in humans.Amusa Buminasan: We want this to be as humanized as possible. These genes are alien in the nuclear genome. You may already have introduced new immune profiles that this alien gene generates. Now, if you want to use xenotropic expression, this will make even more changes. From the point of view of testing, we have to check all the genes of other organisms that have fallen into the nucleus. We can make these changes, but we want the changes to be as humanized as possible.Many of these genes, migrating to the nucleus during evolution, acquired various changes that allowed them to transform. In some animals, Complex I functions due to just one protein, such as NDI1in yeast. But in a human cell, for its functioning, 47 proteins are needed, 7 of them are synthesized from mitochondrial DNA, and the remaining 40 from nuclear DNA.You will not want to express a foreign protein (NDI1), and then try to restore its functionality. From a purely experimental point of view, you can do this, but using it as a therapy in people is not a good idea if you want to maintain the integrity of the entire biochemical complex. I can’t even imagine what difficulties may arise in the process. Gene therapy is complex in itself, and now imagine gene therapy as something like a gene from yeast.Thomas Warner Lehner : What realistic terms for the appearance of human therapy could you name?Amusa Buminasan: Actually, this therapy already exists, but for the transcoded version. That is, we already have a precedent. All we need to do is show that our version is better and has a better immune profile. That's why we want to do it on model animals - to show how much better it is. I can’t name the dates - this is a very difficult question. During the conference, someone already asked me. If animal studies go well, then five years. Not five years before human therapy, but five years to show that the method is quite good, to begin the development of human therapy based on it.Thomas Warner Lehner: There are several factors that favor the accumulation of mutations in mtDNA over time. Is it necessary to supplement the allotopic expression of mtDNA genes with other therapies that will reduce the number of mutations that arise over time in order to see a noticeable effect in aging?Amusa Buminasan : Good question. If you supplement allotopic expression with what you already have, for example, idebenone, elamipretide, or something similar, this will be useful. All of them are antioxidants and improve the function of oxidative phosphorylation .However, it is a question of gene therapy in patients; their illness may not contribute to the adoption of this therapy. Their biochemical complex is not fully functional, but complementary therapy affects only part of the cascade, while mitochondria generally do not function as they should. You may want to correct their disease to a certain level, and only after that apply gene therapy to make sure that gene therapy works.Regarding aging, the debatable question is whether mtDNA mutations are a major factor in aging or not. Our goal is to help patients and, if possible, go further.Thomas Warner Lehner : What, in your opinion, research in anti-aging biotechnology is most needed now in order to advance as much as possible in this area in the next 10 years?Amusa Buminasan : I think we need good biomarkers. This is what is lacking in the field. Everyone wants quick fixes. There are various areas of research, and each researcher considers his own important for the fight against aging, but I do not think that they are all right. I think aging is more like the breakdown of the whole complex over time. Therefore, we need better markers and perhaps a better understanding of what it means to be healthy (in old age). People should not put up with the fact that with age they will age and then die. It may be necessary to draw public attention to the problem so that people understand that it is normal to desire and be able to be healthy throughout life.Thomas Warner Lehner : Which question, of those that you have never been asked before, would you like to hear from journalists?Amusa Buminasan : This is an unexpectedly difficult question. I can not answer it right now. However, I want to say something about MitoMouse . On behalf of MitoTeam at SENS, I want to thank LEAF for their help.ReferencesPfanner, N., Warscheid, B., Wiedemann N. (2019). Mitochondrial proteins: from biogenesis to functional networks. Nature Reviews: Mol Cell Bio, 20, 267-284.
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Translated by Vladislav Genzhera, SENS Volunteers