Turn.bio: Transiently Reprogramming Cells to Near Pluripotence as a Therapy for Aging
Turn.bio is Gary Hudson's latest company, now that others are running the day to day development at Oisin Biotechnologies. The Turn.bio staff are working on a particular take on the idea of inducing pluripotence in cells in vivo as a form of compensatory therapy for aging. This is a concept that struck me as being fairly crazy the first time I saw it discussed in a research publication. It is certainly possible to reliably reprogram somatic cells of near any sort into what are known as induced pluripotent stem cells, capable of differentiating into any type of cell. This is the foundation for the production of arbitrary cell types for transplantation. But doing it inside a living animal? Surely a recipe for cancer and more cancer, as the pluripotent cells replicate uncontrollably outside the normal restraints of a structured tissue.
Oddly, however, the initial outcome in mice was not cancer and more cancer. It was a set of benefits to health and tissue function that looked a lot like the results of stem cell therapies, most likely achieved via the signaling produced by the newly induced pluripotent stem cells. It remains to be seen what the risks look like over the long term, but the result prompted some interest and following studies in the research community. Given this, what if it were possible to guide cells only part-way into a pluripotent state, and only temporarily, generating beneficial signals for a time without any meaningful risk of pluripotent cells floating around in tissues for the long term? That is what the Turn.bio staff are working on. The result may be a more controllable, guided way to achieve the benefits of stem cell therapy without the stem cells. The paper here is the basis for their current development program.
Transient non-integrative nuclear reprogramming promotes multifaceted reversal of aging in human cells
The process of nuclear reprogramming to induced pluripotent Stem cells (iPSCs) is characterized, upon completion, by the resetting of the epigenetic landscape of cells of origin, resulting in reversion of both cellular identity and age to an embryonic-like state. Notably, if the expression of the reprogramming factors is applied only for a short time and then stopped - before the so-called Point of No Return (PNR) - the cells return to the initiating somatic cell state. These observations suggest that if applied for a short enough time (transient reprogramming), the expression of reprogramming factors fails to erase the epigenetic signature defining cell identity; however, it remains unclear whether any substantial and measurable reprogramming of cellular age can be achieved before the PNR and if this can result in any amelioration of cellular function and physiology. To test this, we first evaluated the effect of transient reprogramming on the transcriptome of two distinct cell types - fibroblasts and endothelial cells - from aged human subjects, and we compared it with the transcriptome of the same cell types isolated from young donors.
We utilized a non-integrative reprogramming protocol that we optimized, based on a cocktail of mRNAs expressing OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG (OSKMLN). Our protocol consistently produces induced pluripotent stem cell (iPSC) colonies, regardless of age of the donors, after 12-15 daily transfections; we reasoned that the PNR in our platform occurs at about day 5 of reprogramming, based on the observation that the first detectable expression of endogenous pluripotency-associated lncRNAs occurs at day 5. Therefore, we adopted a transient reprogramming protocol where OSKMLN were daily transfected for four consecutive days, and performed gene expression analysis two days after the interruption.
Analysis of transcriptomic signatures revealed that transient reprogramming triggers a more youthful gene expression profile, while retaining cell identity. Epigenetic clocks based on DNA methylation levels are the most accurate molecular biomarkers of age across tissues and cell types and are predictive of a host of age-related conditions including lifespan. Exogenous expression of canonical reprogramming factors (OSKM) is known to revert the epigenetic age of primary cells to a prenatal state. To test whether transient expression of OSKMLN could reverse the epigenetic clock, we used two epigenetic clocks that apply to human fibroblasts and endothelial cells: Horvath's original pan-tissue epigenetic clock, and the more recent skin and blood clock. According to the pan-tissue epigenetic clock, transient OSKMLN significantly reverted the DNA methylation age.
This data demonstrates that transient expression of OSKMLN can induce a rapid, persistent reversal of cellular age in human cells at the transcriptomic, epigenetic, and cellular levels . Importantly, these data demonstrate that the process of "cellular rejuvenation" - that we name Epigenetic Reprogramming of Aging, or "ERA" - is engaged very early and rapidly in the iPSC reprogramming process. These epigenetic and transcriptional changes occur before any epigenetic reprogramming of cellular identity takes place, a novel finding in the field.
Sarcopenia is an age-related condition that is characterized by loss of muscle mass and force production. We wanted to test whether transient reprogramming of aged muscle stem cells (MuSCs) would improve a cell-based treatment in restoring physiological functions of muscle of older mice. To test this, we first performed electrophysiology to measure tetanic force production in tibialis anterior (TA) muscles isolated from young (4 months) or aged (27 months) immunocompromised mice. We found that TA muscles from aged mice have lower tetanic forces compared to young mice, suggesting an age-related loss of force production. Next, we isolated MuSCs from aged mice (20-24 months). After treating aged MuSCs, we transplanted them into injured TA muscles of aged (27 months) immunocompromised mice. We waited 30 days to give enough time to the transplanted muscles to fully regenerate. We then performed electrophysiology to measure tetanic force production.
Muscles transplanted with untreated aged MuSCs showed forces comparable to untransplanted muscles from aged control mice. Conversely, muscles that received treated aged MuSCs showed tetanic forces comparable to untransplanted muscles from young control mice. These results suggest that transient reprogramming in combination with MuSC-based therapy can restore physiological function of aged muscles to that of youthful muscles.
I'll note that the founders are three of the paper's authors (Prof. Vittorio Sebastiano, Jay Sarkar, and Marco Quarta) while I'm just acting in the CEO role for the moment to get the venture stood up and funded. Those fellows are the heart, soul and brains of the effort. So Turn.bio is their company, not mine.
The key takeaway from the paper is that we can use our rejuvenation cocktail to change gene expression and functional performance of cells from older to younger phenotypes, while never seeing any loss of cellular identity. We can do that easily ex vivo and are working on the in vivo option, as the next step.
Does this involve the use of Yamanaka factors? The paper I've been reading since last fall suggests this will work providing you do not de-differentiate the cells (e.g. reverse them into iPSC's) too much that they "forget" which tissues they are supposed to differentiate into. This is time-based in that you take the Yamanaka factors for a certain number of days and no more (less you get tetratomas everywhere in your body). I've also read there may be ways to do this without using the Yamanaka factors. Call it invivo partial cellular reprogramming. Perhaps Gary can comment on this.
The question is if this can repair the mtDNA as well. If not, then this would have to be supplemented with a therapy to repair that as well. Perhaps a mitochondrial fission/fusion thing.
Very interesting. This one might be many years before practical human therapies but sill is cool. On the other hand, if the signalling factors can be very targeted and temporary this approach can be used in some unexpected places. Say to boost stem cells in heart-attack victims. If your average remaining life expectancy is a couple of years, you will take the remote risk of higher cancer incidence , especially if it can heal the underlying condition.
Is similar to what Dr. Sinclair talked about in the Harvard Gazette?
"SINCLAIR: We're using a combination of Yamanaka factors [used to reprogram differentiated adult cells into induced pluripotent stem cells] that are used to make stem cells currently in a dish, but what we're finding is that you can introduce them into the animal as well. They tolerate it well and tissues rejuvenate.
I haven't published it yet, so I can't say too much, but we're writing up the paper now that shows that parts of the mouse's body that we thought would not ever improve are able to be regenerated. So we're licensing that technology and hoping that it will be tested in the clinic in the next two years".
The cocktail is described in the paper; Turn.bio uses mRNAs representative of the Yamanaka factors but additional factors as well. The time course of applying these factors is crucial to the successful implementation of the treatment.
By the way, it's important to correct a comment Reason makes in his first paragraph: he says that we "...working on a particular take on the idea of inducing pluripotence in cells..." We definitely don't induce pluripotency - the goal is to change cell epigenetic age - not to change cellular identity. Thus we avoid the teratoma risk, and have not seen any teratoma in all proof of concept experiments conducted to date.
Interesting approach - but I feel it is quite far from any human translation
On a different note:
ResTORbio announces transition towards Phase III
Stock drops 22% as they don't have the cash to do it and have to raise money and dilute everyone in the process
Amazing that they are wasting time and investor money for a drug with limited clinical efficacy that big pharma didn't want
How are Turn.Bio getting the muscle stem cells to engraft and survive long term? I thought that this was a major unresolved barrier in stem cell therapies?
Is it due to the cells not being pluripotent stem cells, but rather epigenetically "younger" muscle stem cells? Or is it due to rats and mice having near miraculous powers of regeneration compared to humans anyway?
I'm hoping this research will be replicated in a non human primate soon, as I don't know if the therapies will transfer to primates?
Another cheeky question is - how were the cells transfected ex-vivo? Were Oisin's fusogenix lipid nanoparticles used, or the more standard viral or electroporation?
@BioInvest699 - I think the real reason why restorebio's molecule hasn't been snapped up by big pharma is that there were positive clinical results with low dose off patent standard rapamycin in elderly patients increasing the response to the yearly influenza vaccine.
Even if Restorbio's mtor1 selective molecule gets FDA approval it will face low cost competition from standard rapamycin, maybe severely limiting the price they can charge?
@jimofoz - The method of transfection is described in the paper: "Cells were transfected using either mRNA-In (mTI Global Stem) for fibroblasts and chondrocytes, to reduce cell toxicity, and Lipofectamine MessengerMax (Thermo Fisher) for endothelial cells and MuSCs, which were more difficult to transfect, using manufacturer's protocol."
Hi there! Just a 2 cents. I wish to congratulate the authors.
I too am wondering much about this, it's scary but great at same time; it's scary that after PNR (Point of No Return) we get teratoma formations (in another study they said it was 15 days, here 5 days; so even quicker, might be due to different types of cells having different 'amount of time' before PNR happens for them); thus scary in the sense that all this must be orchestrated to make sure no PNR happens in the other 'quicker PNR' cells, because then teratomas would form there. MAybe, we need a combination of WILT + this (WILT Whole Interdiction of Lengthening of Telomeres), this way cancer odds formation would be reduced for cancers could not highjack telomerase anymore. They would use ALT instead, this would have to also be interdicted. What I worry, as Reason said, is more and more cancer will form (more teratomas...too many and it only takes one to go rogue), will the number be kept low enough that the immune system can (still) do its job. This is scary in that sense. Any error, and cancer would happen, it does not leave much lax/leeway/headroom for error (and errors happen in biogerontology/rejuvenation medecine). 5 days is very precise..imagine on the 6th day, now pass PNR. Pass Point of No Return = Once there, cancer is assured, and that is a form of death sentence (if we can't stop the cancer). Since the others studies said 15 days, and this one is 5 days, it's like 2 weeks or 1 week or so, depneding on the type of cell (most likely iPSCish/stem cellish-like cells
are the ones with the quickest PNR; because they could turn cancerous real quick).
Epigenetic reprogramming is truly the holy grail of biorejuvenation and it's great to see advancement there, I am just cautiously optimistic (because we don't want 'cancer-ladden' mice to happen...); they say that they reversed the epigenetic age (by Horvath DNA methylation clock), but the cell identity is intact/remains (no pluripotency is induced)...but how does this play out...technically speaking to revert a cell to neonatal epigenetic age...is True Age reversal (the cell identity signature has not changed (it seems) but the epigenetic clock tab has). If the past epigenetic studies could make a progeroid mouse live a full/normal life after epigenetic aging reversal, I ask myself, why 'just a normal life'...why not more? Is there a limit to this epiegentic aging back-and-forth (to Rejuvenate a 'already' rejuvenated body....Re-re-re reprogramming of epigenetic clock...is it limiting after repeated each time. If you Reverse the epigenetic age to 0....you are to 0...no matter if you reverse a million times already). Thus, this is equal to true curing death, for death is post-poned forever doing the reprogramming each time back to young epigenetic clock; with that said, I worry of the consequential/cumulative damages/mutations..are they all fixed, I think not, and seeing that the cell identity has not changed...that worries me too; because it is Still An 'OLD' mature cell that has now it's epigentic clock reversed - but how does this affect 'regular aging' of the body; I know that the epigenetic clock and DNA damages can be 2 things, it's why I wonder, what kind of effect would that mean for regular healthy aging/aging to replicative senescence and epidysregulation/epigenetic drifting/DNA methylome changes by time passing (longevity wise). How much would the 'damages' be consequential then...is the replicative 'age' of old cells any longer any important - does it matter that an Aged cell is now a epigentically young cell, in the sense that, this aged 'epigenetically reprogramme' cell is Not a True young cell - it is an Aged Cell that has been reversed and thus still carry its 'identity' (of being old). But how does this translate..I think this may cause more spontaenous mutations because the cell is Still an old cell - that is reprogrammed. It is Not a True yougn cell (identity wise). That's how I see it and from reading the studies, there is ambiguity there about what constitutes an 'aged/old' cell reprogrammed...it can function 'anew' again, but what are the consequences on the body/lifespan. From the study on progeroid mouse, it seems it has a limit, it might not make a mouse live eternally if you reprogramm continuously the mouse's cells. As said, mutations or the fact that these are cells mascarading as young identity cells (camouflaged), they are not young identity but rather epigenetically 'reclocked' on their clocks. That's different. Of course, I am still guessing here. Maybe, it would Not matter, and indeed, all that would matter is simply reverting their epigenetic clock and that is More than sufficient to Completely reverse the Aging program as we know it and sure hope so that it is the case - because if is it IS the Holy Grail we were waiting for, eternal life would now be a possibility (excluding extrinsic factors (death by accidents)). Of course, I am being Ultra optimistic here and they just did this in progeroid mice/normal mouse..do this in long-lived primated/dogs/pigs/long-lived animal and see (making a mouse live 10-25 years would be a signal would we are really coming to that, for the mouse would 10-times longer, just like a naked mole rat living 30 years - it's that that we must accomplish)...
Just a 2 cents.
PS: Waht I also wonder, is why haven't there been researches studies to Counter this terratoma formation - meaning you go right at the matter - and treat it there at the source - epigenetic tinkering Straight in the terratomas, cancers are All about epigenetics/oncoepigenetic and there are specific epigenetic signature/changes that happen long before cancer happens and are precursors to cancer happening (mostly CpG rich islands that become hypermethylated and cause cancer to highjack so many genes/inflammatory pathways for themselves, we have to stop that, on an epigenetic level; this way PNR would be lesser, but I am guessing we are still not there yet and it is more difficulty to stop these kinds of epigenetic transformations that lead to cancer/teratoma). Cancer Can be stopped on epigenetic level because epigenetics orchestrate some of their actions (I guessed it's in that finnicky 'cells have a different identity' upon cancer or transfomration to iPSCs/pluripotency inducing; and thus lose their self and then can become cancerous. It's very tricky this identity thing.
Ex-vivo gene therapy is proving to be pretty expensive and difficult in the CAR-T area. Of course the massive comercial incentive may drive improvements in the automation of ex vivo cell genetic engineering. But an in vivo gene therapy targeted to muscle stem cells with the expression of the factors under the control of an orthogonal small molecule drug like tetracycline could be superior in terms of cost. Give the patient the gene therapy shot into their muscle, then give them tetracycline on 4 consecutive days.
Is there any way Oisin's nanoparticles can be targeted to muscle stem cells? I read about DNA nanoboxes that only open when their Aptamer "latch" encounters a specific cell surface antigen.
Maybe stick Oisin's nanoparticles containing the factors (modified to express only with tetracycline) inside these boxes, and make the boxes aptamer larches open in the presence of a muscle stem cell surface protein. But then this is turning into a bit of a Rube Goldberg device so may not work?
Gary - have you ever thought about sending team tiny trap an email about this?
Gary, if you don't mind answering this, mRNA is great in vitro but surely impractical in an in vivo setting - can you give us any hints how you hope to translate this?
Many Thanks, Mark.
I think you worry too much about terratomas - there is evidence that it is the extracellular signalling that controls what identity the cells finally adopt; so long as the OSKM (NL) treatment is not too long, or the concentration of pluripotent cells created (if they are created) is not too great, cells will integrate with surroundings tissues and differentiate as per the demands of that tissue. Pluripotent cells will not remain pluripotent.
@Mark - all I can say now is that we have seen successful transfection and expression from mRNA in vivo, so we don't anticipate a problem there.
@Jim - I can't get into the details but we're collaborating with Oisin and using LNPs.
Gary. I do not understand why using a complex mixture of Yamanaki factors, which is expensive and unlikely to suit Pharma, when there is a simple way to rejuvenate the cells epigenetically using Δ133p53α + ROCK-inhibition by small molecule?
Take a look:
1. Muhlinen, N., Horikawa, I., Alam, F., et al., & Harris, C. C. (2018). p53 isoforms regulate premature aging in human cells. Oncogene, 37(18), 2379-2393. doi: 10.1038/s41388-017-0101-3 PMC5954431
2. Horikawa, I., Fujita, K., Jenkins, L. M. M., et al., & Harris, C. C. (2014). Autophagic degradation of the inhibitory p53 isoform Δ133p53α as a regulatory mechanism for p53-mediated senescence. Nature communications, 5, 4706. DOI:10.1038/ncomms5706
3. Mondal, A. M., Zhou, H., Horikawa, I., et al., & Liu, X. (2018). Δ133p53α, a natural p53 isoform, contributes to conditional reprogramming and long-term proliferation of primary epithelial cells. Cell death & disease, 9(7), 750. Cell Death & Disease vol. 9, Article number: 750 https://doi.org/10.1038/s41419-018-0767-7 PMC6030220
@ Dzhagarov - what small molecules upregulate Δ133p53α?
For the time being, a small molecule cannot be used to raise the synthesis of Δ133p53α. For this, a vector is needed as for the Yamanaki factors (but much more simple vector). There is also a mechanism by which p53 can, if necessary, activate the synthesis of Δ133p53α.
A small molecule (for example, fasudil or Y-27632) can suppress ROCK. Together Δ133p53α and Rock inhibitor give synergy for rejuvenating of cells.
The research can be used in reversing gray hair? or treat aging skin?. I think those cosmetic conditions are just as important as conditions like Sarcopenia that affects your mobility.
Today's quality of life depends on your looks as much as mobility or even general health(to some extent) and that is the main reason why people feel bad when they grow older and the main reason for ageism in society today.
If it works for beauty and esthetics there's an immediate market for it. Of course, grey hair can be died. Make-up can be put on the skin while the insides are not so easily fixed.
When a woman wear makeup it usually don't hide much of her age. Looking young with makeup is a completely different thing than actually looking young. It can also to be embarrassing for a man to put makeup.
Makeup doesn't change anything while curing the cosmetical signs of aging will have bigger impact on people's lives and society than anything else.
What about using nkx-3 which is commercially available for testing?