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.
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.