Genes in reserve

SENS Research Foundation plans first mouse trials of allotopic expression

Fight Aging!, SENS Research Foundation Scientists Plan their First Mouse Studies for Allotopic Expression of Mitochondrial Genes Translated by Ariel Finerman, Habr

Mitochondria are the "energy stations" of cells, descendants of ancient symbiotic bacteria. They preserved a small fragment of the bacterial genome encoding thirteen genes necessary for the functioning of mitochondria. Most of the other genes passed into the cell nucleus in the process of evolution, as mitochondria became more and more integrated into the cell. Unfortunately, mitochondrial DNA is more susceptible to damage than nuclear DNA, and some forms of damage can lead to mutations and malfunctions of mitochondria. Mutant mitochondria quickly take over the cell, displacing their functional versions in the process of clonal expansion. Then this cell becomes an exporter of free radicals, which leads to a number of age-related pathologies. Peroxide lipids, for example, are the cause of atherosclerosis.

The goal of the MitoSENS research program is to create backups of all mitochondrial genes in the cell nucleus using gene therapy, a process known as allotopic expression. In principle, it will prevent changes in mitochondria by providing a backup source of proteins necessary for their proper functioning. Of course, this is easier said than done. Genes need to be changed so that proteins can migrate to mitochondria, and optimizing insertion and migration is a difficult task. Alas, due to the low level of funding, the work has progressed slowly over the years since allotopic expression was first shown.

Hello everyone This time we have two pieces of news. First, we are preparing a story about a new trick that we have applied to improve the allotopic expression of mitochondrial genes. We are still studying whether we are 100% right before writing a publication and making a statement, but we are very close. Yes, this means that we have performed allotopic expression of more genes. Stay in touch! Secondly, we are at the planning stage of our first test on mice, and we ask you to help us launch it!
It will include testing two technologies that the SENS Research Foundation helped invent: the unique technology of transgenic mice and the application of what we learned in 2016. Our work, we are sure, will show the world that allotopic expression is real, and the future is behind it. In the near future you will see an announcement about a new test. Matthew "Okie" O'Connor, PhD, Head of the MitoSENS Program

Interview with Matthew O'Connor on Longecity

This week we are telling you about the work of the SENS Research Foundation, which deals with the elimination of molecular and cellular damage. Mitochondrial aging is an important part of the human aging process. The SENS program that solves this problem, MitoSENS, is one of the most ambitious and technically complex bioengineering projects. Important events have recently occurred, and you will learn about them in our interview with the head of the MitoSENS program, Dr. Matthew O'Connor.

Longecity: You're on the air!

Matthew O'Connor: Hi, thanks for the invitation!

Longecity: Could you tell us a little bit about MitoSENS?

Matthew O'Connor: Of course. We are developing a gene therapy that corrects mitochondrial mutations. The idea is that mitochondria have their own DNA, their own genes, there are only 13 genes encoding proteins, but they are all important. Problems begin when mutations arise in them, either inherited from your mother, or age-related.

Longecity: And age-related mutations affect almost everyone, right?

Matthew O'Connor: That's right. We are not 100% sure yet, but everything indicates that mitochondrial function decreases with age, and that this is an important aspect of aging that everyone experiences for themselves, for example, in their muscles, as they become weaker with age.

Longecity: At our last meeting, you only had the concept of moving mitochondrial genes into the nucleus. The concept of MitoSENS was to transfer some of these genes to the cell nucleus, in which they would be better protected, and would continue to do their job. What's new with you? Have you moved on from the first two genes you targeted?

Matthew O'Connor: Mitochondrial DNA is more susceptible to damage because mitochondria specialize in creating energy rather than protecting and storing DNA. This is the task of the nucleus in which all our chromosomes live. Mitochondria produce energy, and the byproduct of energy production is free radicals that destroy sensitive DNA. So we tried to insert a backup copy of any of the thirteen genes in the nucleus. You mentioned two that we were working on. We had a publication at the end of 2016 in which we clearly showed that we could take a cell from a patient with mutations in two of the thirteen genes and correct them using our gene therapy.

Longecity: So you were able to correct mitochondrial mutations, restore mitochondrial functions in these cells. Sounds pretty good!

Matthew O'Connor: Yes, it was very clear. We were able to show an increase in mitochondrial energy production, we were able to show oxygen consumption. The reason we breathe, consume oxygen, is that our mitochondria need it to produce energy. We were able to show an improvement in their survival. We were able to grow cells in two different conditions: anaerobically, as cancer cells usually do, or as bacteria grow, and in conditions where they could only survive aerobically if they could consume oxygen using mitochondria. In aerobic conditions, only corrected cells survived, and all mutant cells died.

Longecity: So you had success with those first two genes that you targeted. What about the other 11 genes? Any plans to work with any of them in the near future?

Matthew O'Connor: Yes, actually we are already working on all of them to varying degrees, and I can tell you a little bit about the success. We developed DNA targeting vectors for all 13 protein-coding genes, and we tested them for their ability to produce proteins and direct them to mitochondria. Not all of them work well, and we can't declare victory yet. But we are making progress, and we will report on it soon. We will show which ones work best and which ones work worst. We will talk about the strategies we are working on to improve the continuous process of developing genes and targeting them to mitochondria.

Longecity: I'm not a bioengineer, so could you explain the mechanism by which proteins end up in mitochondria? How does this happen?

Matthew O'Connor: Mitochondria encode only 13 proteins, the nucleus encodes more than a thousand proteins that are transported to the mitochondria. So their transportation is rather a normal process, and the unusual part is protein synthesis in mitochondria. We have studied how the nucleus normally works, and we are trying to change mitochondrial proteins so that they behave like nuclear proteins. The two simplest problems, for example, are that mitochondrial DNA is written in a slightly different language. She still uses the same four letters: A, T, G and C, but the way they are read is slightly different. The first thing we need to do is translate the genes into the language of the nucleus. The second thing we need to do is put a targeting sequence at the beginning of the gene, called mitochondrial targeting sequence or MTS. We take MTS from another gene and insert it at the beginning of any of our 13 genes to target the expressed protein to mitochondria. And we have tested a lot of MTS in our lab.

Longecity: It sounds quite complicated, technically. You have been working on this for several years, what is the biggest challenge in accelerating this potential anti-aging therapy?

Matthew O'Connor: So, the two things I just told you are the relatively easy part, and the difficult part is optimizing the way the code works with MTS and other regulatory sequences that surround the gene, how the gene gets into the genome, how many times it is inserted. There are many different aspects that we are studying, they are the difficult part, including our understanding of how evolution created this system, and figuring out how we can apply it to mitochondrial genes. We are constantly designing and redesigning, trying different changes in genes to try to figure out how to improve their expression, targeting proteins to mitochondria, and then importing into mitochondria, measuring their function.

Longecity: People who follow the research in the field of rejuvenation know that all this is slow, tedious and difficult. Are there any new more effective tools?

Matthew O'Connor: There are two tools that help us. One of them is that in the current era of synthetic biology, you can order any DNA sequence from scratch for just a couple thousand dollars. So nowadays, unlike when I was in graduate school, we can just type the code we want to create on a computer and synthesize it. In the old days, to create a new version, we used a lot of different hacks that took weeks and months, but nowadays you just need to print it and send it by email. This has been a huge boon for us and our ability to test new ideas. The second one is CRISPR, it's not new in molecular biology, but it's new in our project, it allows us to control where in the nuclear genome we insert our genes. It removes the extra variability that engineers used to have to take into account when trying to insert their gene into the genome. This usually happens randomly, in any place, and this is an aspect that can complicate the situation. Nowadays we are starting to control the process by inserting genes more specifically using CRISPR.

Longecity: There are a lot of companies nowadays, they are looking at one aspect of aging, and it seems that some obstacles or unexpected things suddenly appear in their way. I know that you have been very careful in planning how MitoSENS will evolve. For several years, what was the most surprising, or what sudden problems arose?

Matthew O'Connor: One of the problems is that the models of mitochondrial mutations are very limited. For example, I was talking about using CRISPR to specifically alter a gene in the nucleus. But if you want to change the genes in the mitochondria, you can't use CRISPR, it doesn't work in it, or at least no one has figured out how to make it work. Thus, it is impossible to manipulate the mitochondrial genome, which means that no one can create specific mutations in mitochondrial DNA. We use random mutations that occur naturally. In addition, in model systems – for example, such as mice – there are not so many mutations that are usually studied in the laboratory. There are very few mouse mitochondrial diseases, and that's why most of us use humans. This does not mean that we experiment with people, we use human cells. We are limited to cells that are taken from patients who have found rare mitochondrial mutations. And our group is picky about the mutations we want to study because we want specific mutations affecting only one, maybe two genes at a time so we can ask simple questions. Trying to do everything at once is not the best way. I would say that one of the biggest obstacles that slows us down is the lack of good cell lines to work with. We look for them all the time – in publications and at conferences.

Longecity: And the next question: when do you plan to work with whole organisms, and not just with cells in a cup?

Matthew O'Connor: Good question, and I have an encouraging answer. We plan to start raising funds for mouse trials in the coming months. We are writing financial plans. The laboratories promised to grow transgenic mice for us. We have completely designed the mice we need. We found mice with the right mutations. They are not as important as those with which we usually work in cell lines, but with other mice they would not have survived, since mitochondrial mutations are very harmful to health. But we have mice with moderate mutations, and we have already done experiments on their cells, and they are considered to work. Therefore, I think we will have mice soon, but only in a couple of years we will know if we have corrected the mutation. However, we will have mice with our gene probably in less than a year.

Longecity: Sounds great. And the last question: you're working with damaged mitochondria, and the SENS aging theory says, hey, let's just fix the damage and everything will be much better. Do you have any thoughts about current products? Antioxidants like MitoQ or NAD+ precursors, what do you think about them? Do you think they are effective?

Matthew O'Connor: This is a difficult question, because this is not my field, but I will tell you my opinion. I would say that there are some preliminary studies indicating that increasing the level of NAD+ with these dietary supplements can actually have some positive effect on your mitochondrial function. Whether they will improve your health or life expectancy is still an unresolved question. But they could slightly improve energy production. Antioxidants targeting mitochondria are supposedly working, but I don't recommend you running to the shops for them. And yet I think that this area of research is worth paying attention to. The era in which everyone was talking about taking megadoses of vitamin C and E to try to remove all the free radicals produced by mitochondria is gone, since they don't get into your mitochondria. But some seem to fall into them. The problem is that this is a sensitive system, and it is better not to interfere with it. There have been experiments that have shown that some of these targeted antioxidants may be unnecessarily good at removing free radicals and actually damage mitochondrial function. That's why I don't run to the shops for them, but follow the research.

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