Nublar Analysis: the online magazine WIRED recently recapped an exciting stem cell discovery from Dr. Robert Signer's regenerative medicine lab at UC San Diego. Below, we delve deeper into the supporting study and explore how the concepts discussed are related to other breakthroughs in the field of regenerative and longevity science.
In the Chua et al. paper, researchers from the Signer lab describe a novel mechanism by which hematopoietic stem cells (HSCs) regulate protein homeostasis, aka proteostasis. Shared across all cells, this process involves managing rates of protein synthesis, and recycling and eliminating protein waste products. This is particularly important for stem cells, which need to stay lean and clean, effectively avoiding the hoarding of waste products that occurs over time in differentiated somatic cells. Proteostasis involves collecting junk protein aggregates produced as a result of misfolding and mistranslation, and trafficking them to cellular compartments where they’re degraded, typically by the proteosome.
The Virtues Of Accumulating Trash
Singer’s group next showed that healthy stem cells, rather than immediately engaging the shuttling and degradation pathways, sequester the toxic aggregates until they have the bandwidth and/or incentive to address the backlog. Inability to deal with increasing loads of protein waste, even after upregulating proteosome activity, impairs HSC function and self-renewal capacity. Because stem cells have inherently low levels of protein synthesis, they aren’t built to deal with even small increases, rendering them susceptible to disruption of proteostasis.
The team then linked proteostasis to a process known as macroautophagy, which refers to the recycling of non-protein-based macromolecules, organelles, and pathogens by the lysosome or similar degradation compartments. They discovered that HSCs typically have high baseline levels of macroautophagy (aka autophagy), but that it was “plastic”. HSCs can dynamically tune levels of autophagy, and the researchers discovered “bidirectional compensatory crosstalk” between proteosome activity and autophagy.
HSCs can deal with the inhibition of one of these cell cleaning processes, as they seem to be mutually redundant, but knocking them both out causes serious problems to the stem cells’ ability to self-renew, or put another way, stay young. Further, when the rate of autophagy activity, known as ‘autophagic flux’, is reduced, upregulation of proteosome activity tends to age them. As this is antithetical to the nature of stem cells, in situations where proteostasis is overloaded, HSCs pivot to a special kind of autophagy known as aggrephagy, characterized by the shuttling of misfolded and aggregated proteins into specialized degradation compartments called aggresomes, mediated by a protein complex called Bag3.
A compelling finding from this study, as described in the WIRED piece, is that stem cells delay protein processing as a means of resource allocation. Protein recycling isn’t only a waste management program; the resultant free amino acids are utilized for de novo protein synthesis. For progenitor and somatic (more differentiated) cells, the constant need for fuel favors constitutive and immediate proteostasis and canonical autophagy. Stem cells, however, benefit from waiting for the proper regenerative signals to use stored protein resources, particularly as their low levels of protein synthesis don’t typically require quick recycling. They can smartly shuttle protein waste not to the proteosome, but rather to aggresomes for rainy day activation of aggrephagy.
Hallmarks of Aging
As discussed, proteostasis can be perturbed when the cell is overloaded with mistranslated and misfolded proteins, and when unfolded protein response (UPR), proteosome, or lysosome pathways are dysregulated. The accumulation of junk protein aggregates contributes to age-associated phenotypes linked with Alzheimer's disease, Parkinson’s disease, and ALS. ‘Loss of proteostasis’ and ‘disabled macroautophagy’ are two of the twelve Hallmarks of Aging recently updated in López-Otín et al.
The 12 Hallmarks of Aging
Source: López-Otín C et al. Hallmarks of aging: An expanding universe. Cell. 2023; 186(2):243-278.
Genetic manipulation across model organisms such as mice, fruit flies, yeast, and nematodes has demonstrated the ability to shorten and extend lifespan, or to improve age-related dysfunction (e.g., cognition) by manipulating proteostasis. Results from a Phase II clinical trial in ALS supported the notion that modulating UPR could halt disease progression.
Similarly, decreased expression of autophagy-related genes, pathways, and metabolites accelerates aging, and can also be simulated through genetic manipulation. In a mouse model, transcriptional downregulation of the autophagy gene Atg5 lead to premature death caused by multi-organ degeneration. Increases in aging biomarkers such as persistent telomere damage, mitochondrial dysfunction, and the ‘inflamm-aging’ phenotype – marked by increases in IL-6 and TNF – were also observed. Suspension of Atg5 silencing resulted in partial rescue of the aging phenotype, accompanied by reduced inflammation and enhanced immune function. This improvement in baseline health leading to delayed aging was appropriately referred to as “healthspan” extension. A common adage in the Longevity community is that associated interventions seek to add healthy years rather than simply keep an organism alive longer; Longevity's goal is to increase healthspan.
Strikingly, turning Atg5 back on after a period of induced aging resulted in the eventual death of these mice due to the development of tumors earlier and more frequently than controls. The authors postulated that autophagy can act as a tumor suppressor, which when absent can cause genomic instability such as irreversible telomeric damage. Restoring autophagy in these latently malignant cells could help them transform into full-fledged tumors, consistent with the observation made by Singer's lab. In fact, their conclusion that HSC proteostasis is complemented by macroautophagy was based on observing the effects of Atg5 deletion.
Further experiments in mice and fruit flies support increased longevity through restoration or fortification of autophagy, including by applying agents such as spermidine, a pro-autophagic metabolite that could have therapeutic potential. In general, the level of autophagic flux in organisms, including mammals, tends to decline over time, representing one of the pathways by which cellular and systemic aging occurs.
Caraway Therapeutics is developing small molecules to restore lysosomal flux, in line with the hypothesis that neurodegenerative and rare lysosomal storage diseases are caused by dysfunctional cellular clearance of proteins and other macromolecule aggregates. This is in contrast to the amyloid-β targeting monoclonal antibody therapies such as aducanumab and lecanemab, approved for Alzheimer's disease, but which may not address the full picture of disease pathophysiology.
Immune Aging and Inflammation (Inflamm-aging)
Consistent with the concept of immune aging (immuno-senescence), T and B cells in aged organisms show decreased autophagy. These ‘older’ immune mediators have impaired function, inefficiently clearing cells that have collected DNA damage. This results in an accumulation of sick and dysfunctional cells in the body giving rise to inflammation and further disease processes. Linking age-related processes further, disabled macroautophagy hinders the activity of the NLRP3 inflammasome – an innate immune pathway complex tasked with recycling damaged organelles (activated by DAMPs) and clearing pathogens (activated by PAMPs). Conversely, failing to clear damaged organelles via authophagic processes activates the inflammasome, driving cytokine (IL-1β, IL-18) responses associated with inflammatory disease.
Mitochondria-specific macrophagy (mitophagy) and the intertwined role of the inflammasome in cellular aging was covered here.
Practicing Longevity Enhancement
The recognition of proteostasis and autophagy in warding off age-related disease is exemplified by popular behavioral techniques such as caloric restriction. First demonstrated in mice, restricting calories and shortening the feeding window – effectively ‘intermittent fasting’ – extends lifespan. This delayed aging is associated with improvements in underlying processes of age-related diseases such as Alzheimer’s Disease, liver disease, and cardiac dysfunction; and with increases in important disease factors implicated in muscle wasting, neurodegeneration, and oncogenesis. In all these cases, increasing autophagic flux is a driving mechanism of delayed aging.
Conclusive human data will only be available after several decades of ‘running the experiments’, but the community is already validating surrogate biomarkers that support the benefits of this dietary approach to longevity, and which could be used as clinical endpoints in future clinical trials for age-related disease. These include:
Increased autophagy
Decreased inflammation
Lowered fasting glucose and insulin levels
Reduced oxidative stress
Lowered levels of insulin-like growth factor 1 (IGF-1)
Increased sirtuin activity
Indeed, caloric restriction boosts autophagy via inhibition of mTOR and AMPK signaling. If this sounds familiar, it’s because of the recent attention on drugs and supplements acting in these pathways, most notably rapamycin and NAD+ supplementation to restore sirtuins (SIRT1 through SIRT7). The implications are far ranging and mainly focus on restoration of epigenetic regulation, as described by the information theory of aging advanced by the world’s leading Longevity researcher, Dr. David Sinclair, and his lab at Harvard Medical School.
Interventions to Prevent the Stages of Aging
Source: Kane AE and Sinclair DA. Epigenetic changes during aging and their reprogramming potential. Crit Rev Biochem Mol Biol. 2019; 54(1):61-83.
Dr. David A. Sinclair, A.O., Ph.D.
A recent literature review of polycystic ovary syndrome (PCOS) suggests that SIRT1-directed therapy may be superior to current standard of care to alleviate symptoms and potentially to address associated infertility.
Regenerative Medicine in Longevity Science
Regenerative medicine is a major pillar of ongoing longevity research, perhaps best advertised by the $3 B undertaking by Jeff Bezos and Yuri Milner backed Altos Labs, headlined by top names in biopharma such as Hal Barron, Rick Klausner, Jennifer Doudna, Shinya Yamanaka, and Steve Horvath. Despite industry criticisms over the past year that the initial promise of stem cell research has not delivered high yields and diversity of approved therapeutics, or that iPSC-based cell therapy has hit a wall, stem cell programs across diseases and applications are pushing the boundaries of cutting-edge medicine. Hematopoietic stem cell transplant (HSCT) to replace immune cell depletion during the treatment of blood cancers is a well-established application. Notable examples of new applications include:
Biomarkers (VSELs) for early detection of cancer
HSC gene edited therapies for sickle cell disease and β-thalassemia (lovo-cel, exa-cel, etc.)
A leading allogeneic (iPSC-derived) cell therapy program in a pivotal clinical trial (ALLO-501A; ALPHA2)
hESC and iPSC progenitor and differentiated pancreatic islet cells for T1D (VX-880, VCTX-210, SIG-002, Laverock Therapeutics’ miRNA based preclinical program, etc.)
iPSC-derived neuron replacement (ANPD001, ANPD002) and hESC-derived dopaminergic neurons for Parkinson’s (STEM-PD)
PSCs from Lineage Cell Therapeutics for Dry AMD with GA (OpRegen), spinal cord injury (OPC1), and telomerase damage-induced immune response in cancers (VAC2)
Immune-mediated inflammation in ischemic stroke (MultiStem platform from Athersys) and Covid-associated ARDS (Multistem and Pluristem’s PLX-PAD)
The stem cell market is valued at ~$30 B by 2027 (10.2% 5-Yr CAGR; source: Mordor Intelligence) a figure that should pique interest among Pharmas looking to go on a buying spree during a time of arguably highly favorable valuations.
Of course, proof-of-concept is key, and early pipeline planning is marked by tightened risk appetite and strategic re-prioritizations of indications, TAs, and technologies. Meanwhile, the field of longevity science and medicine is laying the groundwork for a new paradigm of Biotech that will initially focus on well-characterized age-related indications.
Cellular Reprogramming
A famous technological milestone in longevity is the discovery of four cellular reprogramming factors, Oct3/4, Sox2, Klf4, and c-Myc – the ‘Yamanaka factors’ or OSKM genes. These genes are used to induce the transformation of differentiated adult cells into pluripotent stem cells, otherwise known as iPSCs. Longevity research is exploring how to safely leverage the OSK set of Yamanaka factors (c-Myc carries high oncogenic risk) for direct cellular age reversal in vivo, although it’s early days.
Dr. Sinclair’s work is directly relevant here as the age reversal his lab observed in mice following OSK therapy is believed to act by restoring epigenetic function, a master regulator of aging that wanes over time, and another Hallmark of Aging: ‘epigenetic alterations’. Advancement of this research to non-human primate models is ongoing as part of a preclinical research program at Life Biosciences, an industry-leading Longevity platform portfolio company and incubator, based in Boston, where Dr. Sinclair serves as co-founder and Director.
NEWS UPDATE: April 23, 2023 Life Biosciences announced data presented at a conference demonstrating successful restoration of visual function in non-human primates through cellular reprogramming in the eye.
One of Life Bio's platform programs is based on inducing partial epigenetic programming by driving expression of the OSK genes (Oct4, Sox2, and Klf4) to rejuvenate cells that have aged and/or accumulated damage. Prior research from Dr. Sinclair's lab (referenced above) showed that this approach reversed aging and restored age-related vision loss in glaucoma mouse models.
Data presented by Dr. Bruce Ksander (HMS) at the annual meeting of the Association for Research in Vision and Ophthalmology (ARVO) made the significant leap from mouse to non-human primate (NHP, i.e., monkeys), a translational drug development model that serves as the "gold standard" for predicting effects in humans. The group led by Dr. Ksander's and Dr. Sinclair's teams (and other researchers associated with Life Bio) recreated an ophthalmic condition called non-arteritic anterior ischemic optic neuropathy (NAION) in monkeys via laser-induced damage. This simulates deterioration and loss of retinal ganglion cells (RGCs) in humans, which leads to diminished visual acuity and progressive vision loss, and for which there are no effective therapies. Life Bio's intravitreal injection of OSK Yamanaka factors improved two crucial measures of disease reversal in the NHP model, both measured against controls:
Restoration of electrophysiologic signaling consistent with restoration of vision (functional improvement)
Increase in healthy neuronal axon bundles in retinal ganglia (pathophysiological improvement)
NAION is the most frequent cause of age-related acute optic neuropathy (swelling around the head of the optic nerve), caused by impaired circulation that generally occurs over time due to RGC loss, most commonly in individuals over the age of 50. In Life Bio's press release, the preclinical observations from mouse and now NHP are highlighted as evidence for therapeutic efficacy via cellular rejuvenation for age-related disease. Dr. Ksander underscored the importance of these results in further disease applications, noting that the OSK therapy's "potential unlocks new opportunities for cellular rejuvenation, not just in NAION but in other ophthalmic diseases that occur as a result of retinal ganglion cell dysfunction as we age."
Life Biosciences' Epigenetic Reprogramming Platform
Source: Life Biosciences website.
Indeed, Life Bio's epigenetic reprogramming platform (the other is autophagy-focused) is producing convincing evidence that their cellular reprogramming approach can turn back the clock on already-aged cells, rendering them functionally younger, and importantly, reversing disease pathology. Altogether, this data represents robust preclinical proof of concept in an advanced animal model (NHP), a reliable model organ (the eye), and an indication class (ophthalmic and ocular diseases) with high unmet need. Life Bio has indicated that these data will support human clinical trials, a major milestone for Longevity medicine.
Another HMS and Boston/Cambridge translational medicine superstar, Dr. George Church, co-founded Rejuvenate Bio. Church's team has explored the delivery of OSK genes via AAV (a leading delivery vehicle in investigational and approved gene therapy) to mice with a corresponding human age of approximately 77 years old. The published findings showed more than doubling of lifespan and improvements in baseline health metrics such as frailty, lending credence to the idea that Longevity seeks to increase healthspan, not only lifespan. Epigenetic changes were also observed in this study, supporting the mechanistic rationale.
Rejuvenate Bio's suite of preclinical assets leverage a different set of putative longevity factors: FGF21, TGFβ1, and αKlotho, familiar to industry as they’ve been studied in the context of age-related diseases such as CKD, cancer, diabetes, and Alzheimer's disease. The company plans to deliver the three factors in a 'gene addition' paradigm via AAV, almost as a ‘longevity gene therapy’. Lead candidate RJB-01 is in IND-enabling stages for cardiac, metabolic, and renal disease, as well as for longevity in animals – another emerging area in which Gallant Therapeutics and Loyal are making strides.
By virtue of addressing the root causes (hallmarks) of aging and delivering multi-morbidity compression, Longevity Biotech provides an entirely new value proposition.
Conventional Biotech and Longevity science are increasingly overlapping for three principal reasons:
Underlying biology and disease pathophysiology of age-related disease is better understood and characterized by existing biomedical frameworks combined with a more refined classification of the Hallmarks of Aging
Technological breakthroughs in Biotech enable targeting of Longevity pathways, e.g., gene therapy / editing, (stem) cell therapy, RNA therapeutics, etc.
Longevity Biotechs are strategically targeting established indications in age-related disease to generate proof-of-concept vs. standard of care (clear regulatory path); these trials aim to incorporate novel surrogate Longevity endpoints
The Targeting Aging with Metformin (TAME) clinical trial, managed by the American Federation of Aging Research (AFAR), is a first in human study to evaluate the effect of a drug on aging as the disease indication.
The trial seeks to demonstrate that the accumulation of disease as we age (multi-morbidity) can be reduced by a single preventative intervention started prior to the emergence of disease pathology (the trial is enrolling healthy individuals 65-80 years old) and administered regularly. Investigators will evaluate whether metformin delays the onset of one or more diseases. The trial will necessarily take many years to complete, but if successful could establish 'aging' as a disease officially recognized by the FDA. More on this in our next blog post.
Dr. Nir Barzilai, MD, AFAR's Scientific Director is leading the TAME trial
Certainly, naysayers abound with regards to the potential, viability, and status of Longevity science and medicine, however the ship has left the port, and Big Pharma is already investing in the space even if not overtly characterizing internal development and M&A as Longevity focused. We're excited to see the convergence of discoveries in regenerative medicine toward acute disease management and long-term health.
Author
Rohan D Gidvani, PhD
Partner, Nublar Consulting
rohan@nublarconsult.com