An Interview with Vittorio Sebastiano of is working on an interesting approach to induction of pluripotency in the tissues of living animals. They use a form of temporary reprogramming to take cells only some of the way to a pluripotent state, far enough that they issue the sort of beneficial signaling expected of induced pluripotent stem cells, and potentially also repair some of their internal damage, such as via the clearance of dysfunctional mitochondria, but not so far they they actually become induced pluripotent stem cells. The cells revert back to their original state, but with the benefit of some damage repair, and a changed signaling environment. As the company progresses, we shall see whether or not this more careful, partial approach is enough to avoid the risk of cancer that is suspected to result from inducing pluripotency in vivo.

We've already seen successful partial cellular reprogramming in living animals through OSKM induction. How does your approach differ?

Well, I think that 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. We 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.

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. 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. 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.

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.

Do you think your technology 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 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 it is shown that inflammation 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.



Just a quick comment: is working on methods to *avoid* induction of pluripotency when reprogramming. It has shown restoration of a youthful phenotype to otherwise aged cells, measured by the Horvath clock, gene expression and proteomics as well as actual restoration of function. That's why it is called particle reprogramming or "ERA (Epigenetic Reprogramming of Aging). But the cells are clearly shown not to be pluripotent. I'd reword the first sentence...

Posted by: Gary at April 8th, 2019 9:32 AM

I think their short-term, ex vivo approach will fail, for the simple reason that reinjecting a load of somatic cells is not going to do much in a particular tissue; what you actually want in this situation is pluripotent stem cells. They are small, they can cross tissue boundaries, and will migrate directly to sites of injury and effect repair. Best of all, if you inject them into the blood stream their concentration and therefore signalling is diluted, so you do not get teratoma formation. Pluripotent stem cells will be directed by their tissue environment to become whatever cells are needed - they will not remain pluripotent.

Bottom line: stop worrying about inducing pluripotency!


Posted by: Mark at April 8th, 2019 9:37 AM

@Mark, lab testing shows that function is definitely restored in murine muscle. So, your fail prediction may be premature.

fyi Methuselah is first investor/funder of Turnbio.

Posted by: David Gobel at April 8th, 2019 10:09 AM

@David, I would like to be wrong, but even if I am - why would you use this technique when ex vivo iPSCs can be used instead, and treat every tissue at the same time, depending on the requirements of the body at the time?

Having said all that, perhaps the in vivo application will have some advantages over inducing iPSCs.

Posted by: Mark at April 8th, 2019 10:12 AM

Looks like Turn.Bio and Oisin are trying to figure out the long-standing gene therapy delivery challenge:

"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."

I wonder what system they can use to prevent their fusogenix particles from binding and entering cells that are not their target type?

Maybe some kind of aptamer embedded in the lipid nanoparticle membrane, whose stem binds to the fusogenix protein, inactivating it, but when the head of the aptamer binds to a cell surface marker protein the stem unbinds from the fusogenix protein, allowing it to fuse the lipid nanoparticle with the target cell?

Posted by: jimofoz at April 8th, 2019 11:11 PM

Lipid nanoparticles ie exosomes - remarkably useful as delivery vehicles eg for mRNA.

Exosomes probably orally bioavailable by the way. Low dose might well equal moderate yet useful effects which could be repeated serially. This appears to be the aim of not too hot and not too cold!

Very good point re aptamers. I was wondering how one might rig it such that the load goes to the preferred target only.

Posted by: JLH at April 9th, 2019 4:18 AM

@JLH: I'm curious why you think exosomes may be orally bioavailable? I've made no real study of them, but this is the first comment I've read that suggested that.

Posted by: Gary at April 9th, 2019 9:03 AM

Very hopeful. Another bullet in the gun to shoot down aging.

Posted by: Zan at April 10th, 2019 12:15 AM

If Oisin can deliver mRNA to cells then we are a much closer to defeating aging than most people think. Imagine what you could do with effective mRNA delivery!

Posted by: Mark at April 11th, 2019 3:29 AM
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