We recently attended the Undoing Aging Conference in Berlin and had the opportunity to interview Professor Vittorio Sebastiano of Turn.Bio, a company developing partial cellular reprogramming techniques to reverse cellular aging.
As we age, our cells experience changes to their epigenetic markers, and this, in turn, changes gene expression, which is proposed to be a primary reason we age. Recently, there has been considerable interest in resetting these epigenetic markers to reverse cellular aging; induced pluripotent stem cell (iPSC) creation uses similar techniques.
However, unlike iPSCs, which are totally repro grammed back to a developmental state and can become any other cell type in the body, the goal of partial cellular reprogramming is to reset the epigenetic aging markers in the cells without erasing cell identity. Researchers believe that exposing aged cells to reprogramming factors only for a very short time may be enough to reset cellular aging without causing the cells to forget their current roles.
Earlier this year, the Turn.Bio team published a study showing the potential of using transient mRNAs to trigger partial cell reprogramming.
Some researchers suggest that epigenetic alterations are a primary reason why we age and others suggest that it’s a consequence of other processes. Which do you consider correct, and why?
My personal opinion is that I can’t really decide whether the epigenetic changes are the cause or the consequence. I cannot decide what theory is right in the sense that some people suggest it’s a developmental program of aging and some people say it’s a consequence of damage accumulating. What I really care about, at the end of the day, is that, regardless, epigenetic changes explain aging. The epigenetic changes are what, at the nuclear level, triggers this dysfunctionality of the cell.
What I also really care about is the fact that epigenetics is really the core of cell behaviors, cell physiology, and so by tackling that aspect, I think we can really tackle the vast majority of the hallmarks of aging.
We’ve already seen successful partial cellular reprogramming in living animals through traveling OSKM induction from Ocampo et al. over at the Salk Institute. How does your approach differ from the direct induction of OSKM using doxycycline that they used?
Well, I think that the Ocampo work is absolutely the first proof of principle that some kind of cellular rejuvenation is triggered by the expression of reprogramming factors. The only caveat is that our work is significantly different from their work, in the sense that our work really demonstrates for the first time that in the naturally aged context, that’s what we can also do. Because if you look at the paper, they use progeroid mice, which are a genetic model of aging, but such a model does not recapitulate the complexity of natural aging. That’s the first point.
The second point is that when they looked at naturally aged mice, the oldest that they looked at was 12 months, which is not a geriatric population but kind of middle-aged. In the context of humans, they used an in vitro tissue culture with induced aging, which, again, is by far not as complex as the aging that you see in vivo over the course of 70 to 80 years.
Our work is fundamentally different; we really looked at human samples all the way from 50 to 95 years old. We have shown this across multiple cell types; we have looked holistically and comprehensively at all the hallmarks of aging, including transcriptomic, methylation clock, physiology of aging, and stem cell homeostasis.
Another fundamental difference is the fact that we’re using mRNAs. Now, mRNAs are non-integrative, they are clinically translatable, and so they huge potential to bring this to the clinic.
You are using Oct4, Sox2, Klf4, and c-Myc (OSKM) but also LIN28 and Nanog to make OSKMLN; what are these two additional factors doing to facilitate cellular reprogramming?
This is the cocktail that we routinely use in my lab for IPS creation, and we have seen that this particular combination is particularly potent and effective at promoting cellular rejuvenation. LIN28 is also linked to the down-regulation of Let-7, which is an mRNA, that promotes differentiation.
My belief is that we’re actually not only working on the epigenetic level but potentially also working on the post-transcriptional level, for example by making sure that the levels of Let-7 get down-regulated to the expression of LIN28, and this actually has an additional effect on the rejuvenation process.
Obviously, there’s still a lot to learn. We’re doing a lot of work in terms of understanding at the molecular level what’s going on. We really want to understand because it is really clear that, to some extent, we can really decouple with two processes of dedifferentiation and de-aging, if such a term exists.
There seems to be something going on in the early phases of reprogramming that actually is taking care of this epigenetic noise or the epigenetic dysregulation that occurs in the cells, particularly in aged cells. So there is really something going on that occurs way before the change in cell identity kicks in. I think that this is going to be really an amazing platform, not only from a therapeutic standpoint but really also in understanding the process of aging per se.
Very interesting, it really is. Cellular reprogramming is probably the approach that I’m most enthusiastic about out of all the approaches people are working on. I think it’s the most exciting for me personally.
I strongly agree with that statement.
Of course, we all have our pet theories and obviously, it’s early days, and only through rigorous testing will we prove or disprove if it will work. I think that the weight of evidence, certainly in the last few years, has moved in support of what Hallmarks of Aging suggested back in 2013, but they lacked the in vivo evidence at the time. That’s why the Ocampo paper for me was a victory moment because it filled in the blanks.
Absolutely. I mean, this was an absolutely pioneering work.
There’s a balance between reversing the epigenetic aging markers of a cell and the loss of cell identity, which would be very bad; we don’t want a brain cell to suddenly start thinking it wants to be a bone cell. In your experiment, you reach a four day transient expression period, using these factors. How did you reach that four-day figure?
It’s not four days for all cell types; it depends on the cell type. If we differentiate cells like fibroblasts and endothelial cells, we use four days, for chondrocytes, three days, and for muscle stem cells, we use two days. This is actually part of the secret of finding the sweet spot, the empirical moment in time just before the point of no return where the cell is becoming partially reprogrammed but has not yet lost its identity.
Now, how do we get there? That’s based on a lot of work that has been going in my lab. My lab is also really focused on iPSCs, and we generate them almost on a daily basis, because it’s an incredibly powerful technology for tissue regeneration and for setting that up for cell and gene therapy and so forth.
We really use the mRNA platform, which I think by far is the most powerful in terms of reprogramming and the safest because of non-integration. We know that during the process, it takes 12-15 days for cells to go all the way back to iPSCs. We know from previous studies that already, by day five, we can see early signs of the activation of genes that are pluripotency-associated very early, not late, in the process. For fibroblasts or endothelial cells, that’s the time when we see these early events, so we want to stop before that because that would potentially trigger or instigate a potential loss of cell identity.
That really leads onto the next question: you mentioned that it’s different for different cell types; how would we systemically treat a human in this manner if different cells need different reprogramming times?
Well, the short answer to that is that we don’t know that yet, and we need to figure that out. I can tell you the way we’re approaching this, particularly on the company side: there is a short-term application, which is most likely going to be the ex vivo approach. The stem cells are going to be isolated from the tissue, rejuvenated in vitro, and then transplanted back. In that type of scenario, we have a uniform population of cells for which we have found this sweet spot so that we can utilize them. Also, because it is done ex vivo, we can make sure the target cells have not changed their identity and are safe. That’s one approach.
The second approach, which is our aspirational approach, is of course in vivo delivery, and you are absolutely right, there is heterogeneity of cell types in vivo. We are partnering up with Oisin Biotechnologies, which is an in vivo delivery platform company, so they have figured out a smart way to actually deliver oligonucleotides including mRNAs in vivo, and, of course, we’re partnering with them in trying to develop a system that, by encoding nanoparticles with cell specific tags or cell specific molecules, we’re going to be able to deliver to different cell types different amounts of mRNAs or to deliver them in a cell-specific manner. Again, there’s still a lot of things to figure out, but we are working on the solution.
A concern that I would offer, based on speaking to a lot of stem cell researchers, is that the aged stem cell niche tends to resist the engraftment of freshly transplanted stem cells, which could be a real problem for your ex vivo approach.
That’s true, but on the other hand, if you are going to very effectively isolate stem cells, for example in the case of sarcopenia, our data strongly suggests that even with a small amount of stem cells rejuvenated, we can rejuvenate the entire tissue, the whole of the muscle. So, potentially, we could really think about isolating a few dozens of cells, rejuvenating them, and then putting them back into different muscles, and, with one treatment, could affect pretty much all the muscles in the body.
This could have a dramatic impact on the elderly population, as they suffer from frailty. Imagine if all of a sudden, the muscles in your body, which are also kind of metabolically active, start behaving in a more youthful fashion; just by transplanting a handful of stem cells, it could have a dramatic effect. So, I’m very optimistic.
An additional idea that I had is actually to start developing this technology first in animals, potentially in pets, and actually start figuring out safety and efficacy in these models. This could actually enable us to develop some important preclinical data that would actually speed up the process. That would allow us to figure out in vivo delivery, and it will also help us figure out what’s going on on the path to human therapy.
The ERA technology, which you use to partially reprogram cells, do you think it has the potential to make systemic rejuvenation in humans a plausible and available prospect in, say, the next 10 to 20 years?
Yes, I strongly believe so, even though at first glance it may seem really difficult, and maybe to some extent impossible, because we naively think about getting everywhere in the body. There is another possibility: what if we could, for example, as we said before for the muscle, what if we can actually target a tissue, or an organ, or a microniche, that actually has a very dramatic systemic effect on its own?
In other words, what if we could, for example, target the hypothalamus? The hypothalamus is one of the main systemic regulators of endocrine functions, and as we heard yesterday in the talk by Dr. Cai, it is shown that inflammation that is going on in the hypothalamus affects the entire body. So, what if we started with the hypothalamus, or what if we started at the endothelium in the body, which is pretty much everywhere in every single vessel? The endothelial cells secrete a lot of pro-inflammatory or anti-inflammatory cytokines, so just on its own, this one tissue could actually have a dramatic, systemic effect.
Again, that’s another way of looking at things; we are really open-minded, and we’re really thinking about different strategies. One day, I’m sure we can get everywhere, but before then, I think we can we can strategically think about how to get something very good without really targeting every single cell in the body.
Another possible top-down approach like this could be the bone marrow stem cell populations. If you could rejuvenate the bone marrow, your entire range of immune and blood cells may improve and could also have a knock-on effect with the thymus, with more thymic progenitors being produced.
Absolutely. I think about these amazing experiments like parabiosis, which, on its own, tells you that there’s something just in the serum that, on its own, is able to have the systemic effect of rejuvenation. You see effects in the brain, in the muscle, and on the skin, so this tells us that maybe there is a first target that we have to go for that could actually broadly affect the health of the entire system.
I think, broadly, it’s a good idea to help people stay healthier for longer, because quite aside from the actual human aspect of reducing suffering, there’s a huge cost savings in health care too. Rather than just keeping somebody alive but sick, there is the possibility of keeping them alive and healthy instead. There is everything to be gained here.
That’s actually one of the most important aspects that I wanted to highlight. In 30 years from now, there’s going to be probably 9 billion people on this planet, 20% of which, so we’re talking about 2 billion people, are going to be 60 or older. Aging is the number one risk factor for cardiovascular disease, cancer, Alzheimer’s disease and so forth. Now, in line with the geroscience hypothesis is that, if you target aging, then maybe you can hit all these diseases at once, because, again, aging is the number one risk factor.
I think we can really make a difference here, and I want to stress the fact that at Turn.Bio, we’re really, really, really mission-oriented. Our mission is to try to extend the lifespan of people but also to increase their healthspan along with that. I also want to acknowledge my partner Jay Sarkar, who is a PhD student in my lab and who is one of the co-founders of the company. Marco Quarta is also a co-founder of the company who helped us with the experimental work with muscle that we published. I really want to thank them, because it’s a group effort, and we are mission oriented.