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SENS Research Foundation Scientists Plan their First Mouse Studies for Allotopic Expression of Mitochondrial Genes

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Posted 21 September 2018 - 05:16 PM

I notice that the lead researcher on the MitoSENS program at the SENS Research Foundation recently gave an interview at Longecity. This work is focused on the prevention of the mitochondrial contribution to degenerative aging, and has been underway for some years. A separate update at the Life Extension Advocacy Foundation, where a crowdfunding event for MitoSENS was organized back in 2015, notes that progress in this work has continued quietly since the last big announcement, and a transition from cell studies to the first mouse studies for that team lies ahead.

Mitochondria are the power plants of the cell, the descendants of ancient symbiotic bacteria. They retain a tiny remnant of the original bacterial genome, encoding thirteen genes essential to mitochondrial function. Most of the other genes moved to the cell nucleus over the course of evolutionary time, as mitochondria became ever more integrated as cellular components. Unfortunately mitochondrial DNA is more vulnerable to damage than nuclear DNA, and some forms of damage can produce malfunctioning mitochondria, faulty because they lack the essential proteins produced by now broken genes. These errant mitochondria can quickly overtake a cell, crowding out their undamaged peers. That cell then becomes a dysfunctional exporter of harmful oxidative molecules, an outcome that contributes to a range of age-related conditions. Oxidized lipids, for example, contribute to the progression of atherosclerosis.

The goal of the MitoSENS research program is to generate backup copies of all mitochondrial genes in the cell nucleus via gene therapy, a process known as allotopic expression. This will in principle prevent damage to mitochondrial DNA from contributing to aging, by providing an additional source of the proteins necessary for correct mitochondrial function. Of course, this is easier said than done, always true in biotechnology. The genes must be altered in ways that allow the proteins to migrate back to mitochondria, and optimizing the insert and the migration so as to produce an acceptable result is an enormous task. There is a huge configuration space of options to explore, with little guidance from historical studies as to where the best results are to be found in that space. This and the low levels of funding for this part of the field are why work has progressed so slowly over the years since allotopic expression was first demonstrated as a proof of principal.

MitoSENS Update September 2018

Hi, everyone! Time for another exciting mitochondrial update. This time, we've got 2 teasers for you. The first is that we're preparing a story about a new trick that we've discovered to improve the allotopic expression of mitochondrial genes. We're still confirming that we're 100% sure that we're right before writing up the manuscript and making an announcement, but we're very close. Yes, that means we're getting it to work on more genes. Stay tuned!

The second is that we're in the late stages of planning our first mitochondrial mouse, and we're going to ask for your help in getting it started! This will involve combining two technologies that SENS Research Foundation helped invent: an advanced transgenic mouse technology and applying what we learned from the 2016 paper to a mouse model that we think will prove to the world that mitochondrial gene therapy is the future. You should see an announcement about the new mouse project soon.

Interview with Dr. Matthew O'Connor

This week we are once again profiling the work at one of Longecity's affiliate labs, the SENS Research Center is leading the charge in the damage theory of aging. One part of the human system that is damaged and declines in function as we age is the cellular mitochondria. The SENS idea to fix this problem, termed MitoSENS, is one of the more ambitious and technically difficult fixes for damaged mitochondria. There have been some significant developments lately and you'll hear about them in this interview with the leader of MitoSENS, Dr. Matthew O'Connor.

I: Welcome to the program!

M: Hi, it is a pleasure to be with you. Thank you.

I: For any first time listeners, could you provide the digest version of MitoSENS?

M: Sure. We have been working on technologies to try to develop a gene therapy for mitochondrial mutations, the idea being that the mitochondria has its own DNA, its own genes, very few of them, only 13 protein coding genes, but they are all important, essential genes. They are a problem when they get mutated, either through an inherited mutation from your mother, or if you develop a mutation with age.

I: And those mutations that develop with age affect pretty much everyone, correct?

M: Exactly. We don't understand it perfectly yet, but all indications are that mitochondrial function decreases with age, and that this is an important aspect of aging that everyone feels and experiences, for example in their muscles, as they get weaker with age.

I: When we last spoke, you were just in a proof of concept stage, trying to move mitochondrial genes in to the nucleus. That's what we're talking about here, the first concept of MitoSENS was to move some of those protein coding genes into the cell nucleus, where they could be protected, and continue to do good work instead of bad work. What is the latest? Have you moved on from the original two genes you were targeting?

M: Yes, so as you point out the mitochondrial DNA is more susceptible to damage because the mitochondria is specialized into making energy, not for protecting and housing DNA. That is the job of the nucleus, which is where all of our chromosomes live. The mitochondria produce energy and the byproduct of energy production is free radicals, which DNA is pretty sensitive to. So we've been working on trying to create a backup copy for any of these thirteen protein coding genes in the nucleus. You raised the two that we had been working on and talking about for a while. We published something on those two at the end of 2016, and so that went very well, we were able to show clearly that we could take the cell that was taken from a patient who had a mutation in two of the thirteen genes, and rescue that mutation by performing our gene therapy in a petri dish of these cells. We could make them behave and survive more like normal cells.

I: So you were able to fix some mitochondrial mutation, rescue some mitochondrial function in those cells. That sounds pretty big.

M: Yes, so it was very clear that by a number of measures. We could show the mitochondrial energy production, we could show the mitochondrial oxygen consumption - the reason that we breathe, that we consume oxygen, is that our mitochondria need it for energy production. We could show that their survival improved. We could grow the cells under two different conditions: the conditions in which the cells could survive without oxygen, growing anaerobically, the way cancer cells usually do, or the way bacteria grow, or we grew them under conditions where they could only survive aerobically, if they could consume oxygen using the mitochondria. Under those conditions only the rescued cells survived, and the mutant cells all died.

I: So you had some success with those first two genes that you were focusing on. What about the other 11 genes? Any plans to work on any of those any time soon?

M: Yes, in fact we are already working on all of them to various extents, and I can tell you a little bit about that. We've made constructs for all of the, meaning we've designed DNA targeting vectors for all 13 of the protein coding genes, and we've tested them to various extents for their ability to produce the gene products to send them to the mitochondria. We've had varying levels of success, so they are not all working sufficiently well yet that we can declare victory and go home. But we will have some new progress to report soon on all of them, I think. We'll show which ones are working best and which ones are working less well. We'll be able to talk about strategies that we are working on to improve the continuous process of engineering these genes for targeting to the mitochondria.

I: Now I'm not a bioengineer, so could you explain the mechanism by which genes that might be sent to the nucleus and encode mitochondrial proteins, how do those proteins end up back at the mitochondria? How does that happen?

M: So the mitochondria only codes 13 proteins, the nucleus codes over a thousand proteins that go to the mitochondria. That is more the normal course of events, whereas the weird part is making proteins in the mitochondria. What we've done is we've studied the way that the nucleus does the job normally, and are trying to adapt the mitochondrial proteins to act more like the nuclear ones. The two simplest components of that are that, for one, the mitochondrial DNA is written in a slightly different language than the nuclear DNA. They still use the same four bases, A, T, C, and G, but the way you read that string of letters is slightly different. The first thing we need to do is to translate that into a language that the nucleus understands. The second thing that we have to do is put a targeting sequence on the front of the gene, and this is called a mitochondrial targeting sequence. We pick one or more to test, and we've tested many in our lab, of these sequences, and move them from a different gene to be in front of any of these 13 mitochondrial genes, and use that to target the product to the mitochondria.

I: That sounds pretty difficult, technically speaking. You've been working on it for a few years now, what is the biggest challenge in terms of speeding along this potential therapy to rejuvenate the human body?

M: Actually, the two things I just laid out are relative the easy part, and the hard part is optimizing the way the code works with the targeting sequence, and then other kinds of regulatory sequences that surround the gene, upstream and downstream of the gene, where the gene goes into the genome, how many times it is inserted. There are a lot of different aspects to this that we are playing with that end up being the difficult part, and understanding how evolution has created this system, and figuring out how we can adapt it to the mitochondrial genes. We are constantly engineering and reengineering, trying different little tweaks to the sequence of these genes, in order to to try to figure out how to improve the production of the gene product, the targeting to the mitochondria, and then the import into the mitochondria, and then measuring whether or not it is behaving functionally.

I: People who follow rejuvenation research, such as the stuff that you are doing, know that it is slow, it is tedious, and this kind of work is very complicated. Are there any new tools that you see arriving on the scene that might help produce results more efficiently?

M: There are two tools that are helping us right now. One is that in the current era of synthetic biology, when we have more and more tools to create new DNA sequences, such that today, it is relatively affordable, in the hundreds to low thousands of dollars, to have a company synthesize any DNA sequence that we want to test just from scratch. So these days, as opposed to when I was in graduate school, we can just type on a computer the code that we want to create, and have it synthesized. In the old days, we had to use a lot of fancy tricks that would take up weeks and months of a scientist's time, to create a new version, but these days it is becoming more and more affordable just to type it out and send it off. That has been a huge boon to us, and our ability to test new ideas. A second one is CRISPR, and this is something new, not to molecular biology, but new to this project, that is allowing us to control where in the nuclear genome we are inserting our sequences. That takes out a variability that traditionally scientists have had to content with, where when you are trying to insert your gene of interest into the genome, usually it goes in randomly, anywhere, and that is an aspect that can complicate things. We are now starting to control this by inserting genes more specifically using CRISPR.

I: Anyone who begins any kind of research project into rejuvenation, there are a lot of companies out there nowadays, they look at one aspect of aging, and it seems all of a sudden there crop up a few roadblocks or unexpected things along the way. I know you've been very careful in planning out how MitoSENS is going to progress. Over the past few years, what has been the most surprising thing, or roadblock that you didn't anticipate?

M: One problem that we have is that the models that are available to study mitochondrial mutations and mitochondrial disease are quite limited. For example, I was just talking about using CRISPR to target nuclear DNA specifically. Now for inserting our sequences, that's great, but if you wanted to target something into the mitochondria, you can't use CRISPR, it doesn't work there, or at least no-one has figured out how to make it work there. So there's no way to manipulate the mitochondrial genome, and that means that no-one can create custom mutations in mitochondrial DNA. We are left with random mutations that occur naturally. Furthermore, there aren't very many models of these mutations in model systems that are usually studied in the labs, like mice. There are very few mouse models of mitochondrial disease available, and so most of us actually use humans. That doesn't mean that we're experimenting on people, but we do use human cells. We are restricted to cells that are collected from patients who have these very rare mitochondrial mutations, and to make that even a little bit more rare, our group is picky about the kind of mutations that we want to study, because we want constrained mutations that only affect one, maybe two genes at a time, so that we can ask and answer simple questions. Trying to do everything all at once turns out to be a messy and careless way of doing things, and doesn't produce results very quickly. I'd say that has been one of the biggest roadblocks slowing us down, a lack of good cell lines to work with. We're always on the lookout in the literature and at conferences for the right kind of cells to work on.

I: That does give me a followup question: when do you anticipate that you will be working with whole organisms rather than just with cells in a petri dish?

M: Great question, and I have an encouraging answer for you. We are planning on launching some fundraising for a mouse project in the coming months. We are writing up funding proposals for this as we speak. We have quotes from a transgenic mouse facility that could produce the mice for us. We have fully designed the mice we want to make. What I said before, that it is rare to find mice that have these mutations, we have found one. It is not as dramatic of a mutation as the ones that we usually work on in the cell lines, but if it was then the mouse probably wouldn't be around to be talking about it, because mitochondrial mutations are so damaging to health. But we have one that does have a mild mutation, and we've already done the experiments on the cells from this mouse, and they are deemed to be working. So I think we're going to have mice fairly soon, but it will be a couple of years before we have progress to report in terms of figuring out whether we've actually rescued the mutation. Nonetheless, we should have mice with our gene in maybe less than a year.

I: That sounds great. A final question here: you work with damaged mitochondria and the SENS theory of aging says, hey, let's just fix the damage and things are going to get a lot better. Do you have any thoughts on a lot of the current products that are out there that people take, supplements that supposedly target mitochondrial function? Antioxidants like MitoQ, or NAD precursors - what do you think about them? Do you think there is much efficacy with some of these supplements?

M: It is a difficult question for me, as it is not my main area of expertise, but I can opine on it a bit. I would say that there is some tentative, encouraging research suggesting that boosting your NAD levels through one or more of these supplements that are available might actually be having some beneficial effects on your mitochondrial function. Whether or not that is going to help you to stay healthy longer or live longer, I think is far from a settled question yet. But they might be modestly boosting mitochondrial energy production. Then the mitochondrially targeted antioxidants I would also say are tentatively encouraging, but I don't want to recommend that people run out and start dosing themselves with it, but I do think it is an area of research worth keeping your eyes on. A generation past, an era in which everyone was talking about taking megadoses of vitamin C and vitamin E to try to soak up all the free radicals being produced by mitochondria, it turns out that those don't get into your mitochondria efficiently, but some of these targeted ones do seem to get into your mitochondria. The hesitation is that this is a sensitive system, that you don't want to mess around with too much. There have been experiments that have shown that some of these targeted antioxidants can do too good of a job and actually end up damaging the function of mitochondria in some of these cases. So I'm going to sit tight before I start taking a lot of these supplements, but I am keeping my eye on the research.

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