Top-Line Intervention Choices

After several months of evaluating a great many candidates according to the criteria described and explained above, we have tentatively landed on the following choices for RMR2. The first four interventions in this section are primarily considered, while the next four (RMR2+) are additional interventions we would like to include, subject to funding limits. These are of particular interest given the promising results reported in the past 12 months by other groups and their potential to provide critical insight about longevity mechanisms and synergies. As described below, some details remain to be determined, in terms of how the intervention will be constructed or administered. This is ongoing; we will keep our options open until funding is complete, but we are committed to moving as fast as possible in order to hasten the achievement of RMR and thence LEV.

13 Deuterated fatty (arachidonic) acids – D-PUFAs

The first intervention we plan to include in RMR2 consists of deuterated fatty acids, which we have been offered free of charge. Lipid peroxidation occurs as a consequence of metabolism and plays a significant role in cellular dysfunction with aging. Free radicals strip electrons from membrane lipids in a cascading fashion, generating lipid peroxides and other harmful byproducts which damage DNA and proteins. Membrane integrity and fluidity are disrupted, resulting in impaired membrane transport and intracellular signaling, as well as damaging mitochondria, leading to the production of more free radicals. Studies have found that this cascade can be inhibited, however, by replacing reactive hydrogens in candidate fatty acids with deuterium atoms, generating deuterated polyunsaturated fatty acids (D- PUFAs). This isotopic reinforcement makes D-PUFAs resistant to reactive oxygen species (ROS)-initiated chain reactions, allowing them to withstand oxidative damage. Furthermore, it has been demonstrated that the presence of even a small fraction of D-PUFAs among natural PUFAs in membranes will effectively inhibit lipid peroxidation, alleviating disease phenotypes several disease models. Several clinical trials utilizing D-PUFAs have been conducted in humans for a diverse range of pathologies, particularly for cognition and memory, and safety is well-established. Further, D-PUFAs can be provided in animal chow, eliminating unnecessary injections and associated stress on the animals. When consumed, D-PUFAs incorporate into membranes in many tissues, without any reports of toxicity.

Mouse serum albumin - rMSA

Although replicating heterochronic parabiosis or plasma dilution for our study is not feasible, there is promising evidence that monthly administration of virgin albumin in saline is similarly capable of improving multiple healthspan metrics in aging mice and can increase both mean and maximum lifespan.

The considerations being weighed in this case are 1) cost of dosing every 3-4 weeks 2) cost of material synthesis and 3) duration of benefit in animals. While constitutive gene therapy-mediated overexpression might technically be possible, it has not, to our knowledge, ever been attempted outside of defect rescue, and it is likely that continuous overproduction of the protein would be detrimental. Any newly-designed construct would need to undergo some relatively time-consuming validation in vivo, though long-term efficacy would still remain unknown, as the system would be incomparable to previous studies.

We are currently having early discussions with manufacturers to discuss the costs and timeline of producing physiochemically virgin mouse serum albumin at scale in Pichia pastoris, a yeast expression system which is highly effective for producing pharmaceutical quality heterologous proteins for therapeutics, particularly for proteins which are glycosylated, secreted, and require proper folding. Like any foreign product given to research animals, it must be of high quality and suitable for animal use. Our current estimates for albumin manufacturing are optimistic – currently relying on manufacturers’ ability to inversely scale cost with bulk production.

Mesenchymal stem cells or Exosomes

The progressive loss of stem cell regenerative potential remains one of the most obvious consequences of aging and is a primary focus of rejuvenation therapeutics. Thus, therapies to restore stem cell functionality, including stem cell transplant, are promising strategies for longevity medicine. Stem cell aging remains a high-value target for rejuvenation therapeutics, particularly those aiming for a systemic benefit with possible lifespan extension. Therapeutic administration of stem cells is already demonstrated to improve disease and aging phenotypes in animals and in humans. Our first RMR study (RMR1) also included youthful stem cells as an intervention, however with some key differences, mainly in that it utilized lineage-depleted bone marrow stem cells (HSCs) isolated from young mice. While HSCs populate the cells of the blood and immune system, MSCs constitute an important part of the BM microenvironment that houses HSCs. In addition, the MSC lineage gives rise to many tissues including bone, fat, muscle, and cartilage, as well as endodermal and ectodermal tissues such as neurons, blood vessels, skin, and cells of the liver, pancreas, heart. In this regard, one benefit of MSCs is their relative abundance in the body, as they are enriched in the BM as well as in adipose tissue, skin, muscle, dental pulp, and birth-associated tissues including placenta, umbilical cord, and amniotic fluid and membranes, among other tissues, including peripheral blood. They have been isolated from every mouse tissue and are believed to reside in all postnatal organs.

One possibility being studied to enhance MSC benefit is niche rejuvenation, which could be coadministered with MSC treatment. One recent study identified NTN1 as a molecule which supports a youthful stem cell environment, and found that donor cells had higher engraftment rates and restorative action in old mice when mice were coadministered supplemental NTN1 We are actively exploring the feasibility of this enhancement, but it is a decision we can make at a late stage. Importantly, of stem cell transplant studies in clinical trials, those which have shown the greatest success utilize freshly-isolated cells, as opposed to cryopreserved or culture-expanded. The same is true for HSCs, which is why we opted to increase the project cost by at least $500k (mostly manpower costs; described earlier) in order to avoid freezing.

Another option may be to employ “induced MSCs” (iMSCs) derived from iPSCs Since the first report of this approach is very recent (published in 2023), we are still evaluating it. Intravenously administered MSCs have not been shown to integrate into recipient tissues, and rejuvenating effects are generally attributed to secretory factors which may act locally and interact with the immune system. This motivates consideration of ways to increase the half-life of MSCs in the circulation, and we are currently evaluating a potential approach to that. A word should be added here about why we are choosing to administer MSCs rather than the exosomes they generate, which are also indicated to have therapeutic benefit. A number of features argue against exosomes. First, exosomes are very challenging to characterize, and are typically very heterogeneous mixtures. Contents are often unknown, and differ depending on species, age, tissue type, and normal physiology. Thus, obtaining a standardized mixture is particularly challenging. Another drawback to this approach is the durability of benefit. Although it is believed that the vast majority of systemically administered stem cells are eliminated from the system within a few days of injection, there are still significant and much longer-lasting physiological changes which result from body’s response to cell injections, which are not expected to occur with exosome treatment. It seems likely that the benefits of MSC therapy are via the ability of transplanted cells to induce changes in resident cells, promoting the switch to a regenerative phenotype, which further rejuvenates cells and tissues downstream. Some of these induced changes are not expected to occur without the cells themselves, thereby limiting the extent of tissue rejuvenation which can be achieved with exosomes alone.

Partial cellular reprogramming

Partial reprogramming has attracted substantial interest in recent years both from a research and investment standpoint due to its potential for rejuvenation extending beyond individual cells to affect entire tissues and organ systems. The process begins with rapid metabolic changes, as cells shift toward more youthful energy utilization patterns. This metabolic remodeling is crucial, as it provides the necessary substrates and energy for subsequent rejuvenation processes while simultaneously influencing epigenetic modifications through metabolite availability.

As these initial metabolic changes take hold, they trigger widespread epigenetic remodeling. Key metabolites like NAD+ and α-ketoglutarate serve as essential cofactors for epigenetic enzymes, enabling the restoration of youthful DNA methylation patterns and histone modifications. This epigenetic rejuvenation then feeds back to enhance metabolic function by modulating the expression of metabolic genes, creating a self-reinforcing loop. Simultaneously, improved energy availability and epigenetic remodeling together enhance cellular protein quality control systems, leading to better clearance of damaged proteins and improved cellular function.

The coordination between these fundamental processes - metabolism, epigenetics, and proteostasis - creates a robust foundation for tissue-specific rejuvenation. In muscle tissue, for example, these changes manifest as improved contractile function and better energy utilization. In neurons, they support enhanced synaptic plasticity and neurotransmission. Hepatocytes show improved metabolic and detoxification capabilities. These tissue-specific improvements, in turn, contribute to systemic benefits through enhanced organ function and improved inter-tissue communication.

The systemic nature of rejuvenation is further amplified by the interconnected stress response pathways that are activated during partial reprogramming. These pathways, including the heat shock response, unfolded protein response, and DNA damage response, work together to enhance cellular resilience across tissues. Key regulatory hubs like mTOR, sirtuins, and FOXO factors integrate these various processes, ensuring coordinated responses throughout the organism. This molecular orchestra creates a comprehensive rejuvenation program that can restore youthful function at multiple biological scales - from individual cells to entire organ systems.

The most responsive cell types to partial reprogramming tend to be those with high metabolic activity and critical regulatory functions, creating cascading benefits throughout the organism. Skeletal muscle cells, for instance, show particularly robust responses, with enhanced mitochondrial function, improved force generation, and better metabolic regulation. This muscle rejuvenation extends beyond mere physical strength - it influences whole-body metabolism through improved glucose handling and myokine secretion, potentially affecting systemic aging processes.

Neurons represent another highly responsive cell type, with partial reprogramming enhancing their plasticity, metabolic efficiency, and synaptic maintenance. The rejuvenation of neuronal populations, particularly in regions like the hippocampus and hypothalamus, may have far-reaching effects on longevity through improved cognitive function and better neuroendocrine regulation. The hypothalamic-mediated changes can affect everything from energy metabolism to immune function, creating organism-wide benefits.

The liver's high responsiveness to partial reprogramming is particularly significant for longevity. Rejuvenated hepatocytes show enhanced metabolic flexibility, improved protein synthesis, and better toxin clearance. These improvements affect the entire organism through better regulation of blood glucose, more efficient protein homeostasis, and enhanced detoxification capacity. The liver's central role in metabolic regulation means these improvements can significantly impact overall health span.

Stem cell populations across various tissues also show marked improvements with partial reprogramming. Enhanced stem cell function in bone marrow, muscle, and other tissues improves tissue maintenance and repair capacity. This improved regenerative potential could help maintain organ function with age, potentially extending both lifespan and healthspan. The rejuvenation of stem cell niches may be particularly important, as these microenvironments influence stem cell behavior and tissue homeostasis.

Achieving efficient and safe delivery of reprogramming factors to specific cells or tissues in vivo, however, still presents a considerable challenge and the development of practical, targeted, and cost-effective delivery methods is vital for successful application. The delivery of these factors has historically been achieved using viral vectors or genetic modifications, however recent innovations have focused on liposome-mediated delivery as mRNA, and even chemical induction of reprogramming factors using reagents and small molecules.

Anti-IL-11

The pro-inflammatory cytokine IL-11 is increasingly recognized as a significant component of the senescence-associated secretory phenotype (SASP) and has emerged as a promising longevity target due to its central role in age-related fibrosis and inflammation across multiple tissues. IL-11 production is upregulated in response to oxidative stress as a compensatory mechanism, yet sustained IL-11 activity paradoxically worsens tissue damage by promoting reactive oxygen species (ROS) production. This accumulation of ROS leads to cellular senescence, a condition where cells lose the ability to divide and repair tissue effectively. In the liver, for example, increased IL-11 exacerbates oxidative stress, aggravating liver fibrosis and reducing regenerative capacity (Nishina et al., 2012). In mice, genetic deletion or pharmacological inhibition of IL-11 signaling has recently demonstrated remarkable therapeutic effects in key organs that typically deteriorate with age. In the heart, IL-11 inhibition prevents and reverses cardiac fibrosis by blocking myofibroblast activation and reducing extracellular matrix deposition, ultimately preserving cardiac function. Similarly, in the liver, disrupting IL-11 signaling reduces hepatic stellate cell activation and fibrosis, while improving metabolic parameters and glucose homeostasis. These effects appear to be mediated through the interruption of ERK/MAPK and STAT3 signaling pathways, which are key drivers of cellular senescence and tissue dysfunction. (Schafer et al., 2017; Chen et al., 2020).

The therapeutic potential extends beyond individual organs, as IL-11 inhibition shows systemic benefits through its effects on stromal cells and the broader inflammatory environment. For example, increased IL-11 with age dysregulates immune responses by stimulating persistent inflammation, which causes immune cells to infiltrate tissues. This inflammatory environment damages tissues and alters the normal healing process, resulting in a pro-fibrotic state rather than resolution. In cardiovascular tissue, for instance, this persistent inflammation underpins the development of age-related heart failure and other cardiovascular diseases (Xu et al., 2002). In adipose tissue, reduced IL-11 signaling decreases inflammation and improves metabolic health, while in skeletal muscle, it may help maintain tissue integrity during aging. Human studies have revealed increased IL-11 expression in various age-related pathologies, including heart failure, liver cirrhosis, and chronic inflammatory conditions, suggesting strong translational relevance. The broad tissue distribution of IL-11 and its signaling components, combined with its role in fundamental aging processes like fibrosis and inflammation, positions IL-11 inhibition as a potentially powerful intervention for extending healthspan. Research in aged animal models indicates that IL-11 inhibition not only improves tissue- specific functions but may also contribute to lifespan extension. By addressing chronic inflammation, reducing fibrotic progression, and improving overall tissue health, IL-11 blockers have shown promise in increasing healthspan and potentially extending lifespan (Widjaja et al., 2024). The fact that mice lacking IL-11 signaling show improved health outcomes across multiple organ systems suggests that targeting this pathway could offer comprehensive protection against age-related decline.

CDC42 inhibition – CASIN

Elevation of Cdc42, a small RhoGTPase, plays a significant role in the aging process by disrupting cellular functions essential for maintaining tissue homeostasis. As individuals age, Cdc42 activity naturally increases in various cell types, including hematopoietic stem cells (HSCs), mesenchymal stem cells, and intestinal epithelial cells. This elevation contributes to functional declines in cell populations critical for regeneration and repair. For instance, in HSCs, increased Cdc42 levels lead to decreased regenerative capacity and cellular exhaustion, which weakens the immune system’s ability to respond to pathogens and is a core contributor to immunosenescence in older adults (Geiger et al., 2007; Florian et al., 2020, Wiley). The causation of Cdc42 elevation in aging is associated with intrinsic cellular signals and age-related changes in the cellular microenvironment. Factors like oxidative stress, altered lipid composition in cell membranes, and shifts in the cytokine milieu with age contribute to amplifying Cdc42 activity. For example, the chronic inflammatory state of aging, known as “inflammaging,” promotes increased Cdc42 activity, leading to the production of pro-inflammatory cytokines and accelerating tissue degeneration (Ito et al., 2014, PLOS One; Wang et al., 2007, PNAS). Such effects can disrupt insulin and leptin signaling, exacerbating age-related metabolic disorders such as obesity and type 2 diabetes (Umbayev et al., 2023, MDPI). The pathophysiological implications of elevated Cdc42 extend across various systems. In tissues dependent on precise cellular architecture, like neural and epithelial systems, Cdc42-induced disruptions in cell polarity can lead to structural disorganization and functional decline. Elevated Cdc42 levels accelerate aging in HSCs, impairing blood formation and immune responses, while also contributing to chronic inflammation that further degrades tissues like the skin and bone marrow (Geiger et al., 2007, Taylor & Francis; Nalapareddy et al., 2021, Cell). These findings underscore Cdc42 as a key player in degenerative diseases linked to aging, making it a prime therapeutic target for age-related pathologies. Thus, inhibiting Cdc42 activity offers potential benefits, especially with compounds like CASIN, a small molecule inhibitor. CASIN has shown promise in preclinical studies by reducing Cdc42 activity, which has restored functionality in aged HSCs, enhanced their regenerative capacity, and decreased systemic inflammation (Florian et al., 2012, Cell Stem Cell). Such inhibition not only reduces senescence markers but also addresses inflammaging at its cellular root, offering a strategy to potentially extend healthy lifespan by preserving tissue function, improving immune responses, and reducing age-related degenerative processes. CASIN and other Cdc42 inhibitors thus highlight a promising approach to rejuvenating stem cell populations and addressing aging’s systemic impacts at the molecular level (Florian et al., 2020, Wiley).

Senolysis (Rockfish)

The potential senolytic mechanism of long chain fatty acid CoA ligase inhibition is rooted in the distinct metabolic vulnerabilities of senescent cells. These cells demonstrate markedly elevated levels of lysophosphatidylcholine and free arachidonic acid, similar to the lipid profile seen in ferroptotic cells - a connection particularly relevant given recent evidence that senescent cells show increased sensitivity to ferroptosis inducers. The accumulation of these bioactive lipids suggests compromised membrane homeostasis, which has been demonstrated to correlate with increased sensitivity to additional membrane stress in multiple models of cellular senescence.

By inhibiting long chain fatty acid CoA ligase, we would prevent the activation of free fatty acids to their CoA derivatives, blocking their incorporation into phospholipids and their entry into beta-oxidation. This would be particularly devastating for senescent cells, which already show impaired lipid homeostasis and increased membrane permeability. The mechanism is analogous to the demonstrated senolytic activity of dasatinib, which disrupts membrane integrity, but potentially more selective due to the pre- existing lipid abnormalities in senescent cells. Supporting this approach, recent studies have shown that senescent cells exhibit reduced expression of membrane repair proteins and decreased capacity to handle acute membrane stress. The combinatorial effect of existing lysoPC-mediated membrane disruption, elevated free AA levels, and blocked fatty acid metabolism would likely exceed the survival threshold specifically in senescent cells, while normal cells could maintain viability through intact compensatory mechanisms and baseline membrane stability.

Oxytocin Therapy

Oxytocin therapy has emerged as a promising intervention for extending lifespan and promoting rejuvenation in various animal models, with studies consistently highlighting its capacity to reverse age- related decline and restore function across multiple systems, including the muscular, hepatic, skeletal, and nervous systems.

Lifespan Extension: In aged mice, oxytocin treatment has been linked to increased healthspan and lifespan through systemic rejuvenation mechanisms. Research by Díaz-del Cerro et al. (2022) demonstrated that oxytocin improves homeostatic regulation and reduces inflammation, key factors in extending healthspan. Mice treated with oxytocin exhibited improved metabolic health and resilience, delaying the onset of age-related conditions (Díaz-del Cerro et al., 2022).

Muscle Regeneration: Oxytocin enhances the regenerative capacity of aged muscle by activating muscle satellite cells. Erdman (2021) showed that systemic administration of oxytocin in aged mice restored muscle repair to youthful levels, mediated by the activation of the MAPK/ERK pathway. This pathway promotes the proliferation and differentiation of muscle progenitor cells, enabling efficient tissue repair and reducing muscle atrophy (Erdman, 2021).

Hepatic and Bone Rejuvenation: Oxytocin therapy has also shown promise in restoring liver and bone health. Zhai et al. (2021) found that oxytocin promotes liver regeneration by increasing hepatocyte proliferation via STAT3 signaling. Similarly, Fernandes-Breitenbach et al. (2022) observed improvements in bone density and strength in aging rats treated with oxytocin. The hormone stimulated osteoblast activity and reduced bone resorption, thereby reversing age-related osteoporosis (Zhai et al., 2021; Fernandes-Breitenbach et al., 2022).

Cognitive and Neuroprotective Benefits

Oxytocin has been shown to enhance brain function and protect against neurodegeneration in aged rodents. Studies by Carter and Kingsbury (2022) demonstrated that oxytocin increases neurogenesis and synaptic plasticity while reducing neuroinflammation. These effects, mediated through the upregulation of brain-derived neurotrophic factor (BDNF), suggest that oxytocin supports cognitive health during aging (Carter & Kingsbury, 2022).

• Molecular mechanisms underpinning oxytocin’s rejuvenative effects include activation of MAPK/ERK and STAT3 signaling pathways, reduction of oxidative stress, and modulation of inflammatory cytokines. Typical therapeutic regimens involve subcutaneous or intraperitoneal administration at doses of 0.5–2 mg/kg/day over periods ranging from days to weeks, depending on the target system.

Potential Interactions

D-PUFAs + rapamycin o D-AA protects membranes while mTOR inhibition enhances autophagy Potential synergies:

• Enhanced mitochondrial quality control

• Improved protein homeostasis

• Better cellular recycling

• Reduced inflammatory signaling

D-PUFAs + senolysis by arachidonic acid buildup

Deuteration slows AA metabolism, in turn retarding the generation of pro-inflammatory eicosanoids while maintaining membrane structural properties and signaling functions. In combination with an AA- mediated senolytic, cellular responses become quite complex and challenging to predict. The simultaneous administration of d-AA and blockade of long chain fatty acid CoA ligase may create a unique metabolic situation where neither regular AA nor d-AA can be effectively activated to their CoA forms, leading to accumulation of unesterified fatty acids, potentially disrupting membrane organization and cellular signaling pathways. While the deuteration still protects against oxidative metabolism where it occurs, many of the beneficial effects of d-AA might be compromised due to the inability to properly incorporate it into cellular lipids. This combination therefore presents a biochemical paradox where the protective effects of d-AA could be overshadowed by the cellular stress of accumulated free fatty acids and disrupted lipid homeostasis. These effects, however, could very well be dose-dependent, as senescent cells are predicted to be uniquely vulnerable to CoA ligase blockade, sparing healthy cells and tissues at low doses, thus opening the possibility that the combination of interventions potentiates the rejuvenation effects of both. The only way to determine if outcomes are synergistic or antagonistic is to combine the interventions and testing dose responses.

Reprogramming + rMSA

One possible reason for the systemic rejuvenation possible with partial reprogramming is due to its ability to restore normal gene expression patterns for critical pathways which become disrupted with age. For example, changes to serum albumin synthesis, structure, and function are well-documented in aging, with transcriptional changes attributed to altered promoter methylation in hepatic cells. Similarly, methylation changes are one reason for global reduction in the expression and activity of antioxidant enzymes in aging, which, in combination with the reduced capacity of albumin, results in doubly diminished cellular protection against free radicals. Because oxidative stresses accumulate so readily with age causing damage to numerous cellular structures, therapeutic combinations increasing antioxidant capacity through different mechanisms may yield significant synergistic benefits. This includes other interventions currently in consideration, such as D-PUFAs to reduce lipid peroxidation, senolysis to eliminate malfunctioning cells generating increased ROS, IL-11 inhibition to counteract fibrosis and restore normal tissue function, which in turn, lowers the production of pro- inflammatory factors and ROS production, etc.

“Baseline” treatments

Combination therapies are only valuable if their benefit exceeds that of the best known alternative. To date, the most effective rejuvenation treatments are rapamycin, caloric restriction, and exercise. We carefully considered these in the context of RMR1, opting to include rapamycin as one of the four interventions for comparison. For RMR2, we are considering giving rapamycin to ALL the animals, i.e. as a baseline intervention without untreated controls. This would allow us to gauge the efficacy of other rejuvenation interventions when the overall damage burden is already slightly lowered.

Similarly, we have determined that animals in the RMR2 study will have access to a running wheel in their cages, permitting voluntary exercise. While the animals in RMR1 are provided some enrichment such as nesting material, wheels are not standard in conventional rodent housing. Physical activity is known to be a strong determinant of healthspan in both animals and humans, and we believe that no intervention can be maximally effective in obese, inactive mice. We do not consider this addition to be an “intervention” in itself, but rather a basic requirement in order to delay aging pathologies.