Robust Mouse Rejuvenation

Study 2

Overview

LEV Foundation's flagship research program is a sequence of large mouse lifespan studies, each involving the administration of (various subsets of) at least four interventions that have, individually, shown promise in others' hands in extending mean and maximum mouse lifespan and healthspan.

We focus on interventions that have shown efficacy when begun only after the mice have reached half their typical life expectancy, and mostly on those that specifically repair some category of accumulating, eventually pathogenic, molecular or cellular damage.

The first study in this program began in early 2023. The second, described on this page, will begin - subject to funding - in 2024.

Goals and Motivations

As in RMR1, the ambition for RMR2 is to achieve "Robust Mouse Rejuvenation". We define this as an intervention or treatment program that:

  • is applied to mice of a strain with a well-documented mean lifespan of at least 30 months

  • is initiated at around 12 months younger than the mean lifespan

  • increases both mean and maximum lifespan by at least 12 months

The primary endpoint for the study is to determine the interactions between the various interventions, as revealed by differences between treatment groups (receiving different subsets of the interventions), on overall lifespan.

However, we are also investigating aging and morbidity trajectories, causes of death, and functional decline. In this way we will add greatly to the understanding of which benefits these interventions confer and how they synergize, or possibly antagonize.

Study Design

Provider: Studies will be conducted at Ichor Life Sciences (Syracuse, NY). Ichor has a long-standing reputation conducting animal studies for pharma and the biotech industry, and has experience performing lifespan studies in mice. They are well-equipped for a study of this size and we know them well.

Age at study initiation: As in RMR1, interventions will begin in mid-late life, between 18-20 months of age, in order to assess the repair/rejuvenation capacity of interventions. The study will run through the remaining lifespan of all mice with the exception of animals selected for cross-sectional analysis at timepoints, as in RMR1.

Mouse Strain: RMR2 will utilize either CL57BL/6J mice or alternatively, we may switch to HET3, which are now available pre-aged through Jackson Laboratories. We are evaluating the benefits and drawbacks of both these strains and will update when a decision has been reached.

Treatment Groups: RMR2 is planned to include 10 groups, as in RMR1, including groups receiving just one intervention so as to validate that we are successfully recapitulating effects reported in prior work. We continue to reason that very little additional information would result from also including the six possible combinations of two out of four interventions. Three out of four, on the other hand, gives key information, especially on the existence of any antagonistic interactions. Finally, the tenth group will be receiving all four study interventions.

Scale of study: We aim to conduct RMR2 on a similar scale as RMR1, including 500 male and 500 female mice. In the event of funding limitations, we may alternatively opt to conduct RMR2 in only one sex, which cuts the study size in half, while maintaining statistical power for individual treatment groups. While this will enable us to initiate RMR2 more expediently, using a single sex remains suboptimal due to significant known sexual dimorphism in mouse lifespan studies.

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 in all treatment groups. 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.

Data Collection: We intend to collect laboratory and functional data longitudinally, as RMR1, in addition to hematological and tissue data cross-sectionally from culled animals and from those humanely euthanized during the course of the study.

We are keen to establish collaborations with academics and industry researchers to take full advantage of the information potential arising from the study and the expertise of the research community. If you are interested in biospecimen or data from RMR2 for your field of study, please contact us at science@levf.org to discuss further.

Interventions

  • 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.

  • Serum albumin is the most abundant circulating protein in mammalian plasma, accounting for approximately 60% of total blood protein. It has a critical role in maintaining the blood’s osmotic pressure and additionally serves as an important carrier protein for endogenous and exogenous ligands such as fatty acids, metal ions and drugs. It is, however, the third main function of serum albumin we are primarily interested in – that is, its role in the maintenance of intravascular redox homeostasis, a property dependent on the redox state of a free thiol on Cys34. Due to serum albumin’s abundance in plasma, this thiol contributes a large amount of ROS scavenging activity when in its reduced state, and changes in the percentage of reduced vs oxidized serum albumin are indicative in states of liver disease, renal dysfunction, and diabetes mellitus, as well as in aging.

    There is promising evidence that repeated administration of physiochemically virgin serum albumin in saline can improve multiple healthspan metrics in aging mice, influencing both mean and maximum lifespan. In addition to bolstering redox buffering capacity, treatment in this way may also confer a plasma-diluting effect, which is known to rejuvenate multiple organs and tissues on its own.

  • 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 and is the focus of ongoing clinical trials.

    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. It seems likely that the benefits of MSC therapy are via the ability of administered cells to induce changes in resident cells, promoting the switch to a regenerative phenotype, which further rejuvenates cells and tissues downstream.

    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. MSCs can be derived from a variety of sources and can be reliably expanded ex vivo, permitting their use at scale and under repeat-dosing conditions.

  • Partial reprogramming involves the temporary activation of a set of genes known as the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), which can induce a state of cellular rejuvenation without altering the cell's original identity. This approach addresses the aging process at the cellular level, potentially complementing other interventions and providing a comprehensive strategy for age-related therapies.

    The Yamanaka factors work by rewiring the cellular epigenome, erasing certain age-associated epigenetic marks and activating genes associated with youthful characteristics. This leads to functional rejuvenation in various tissues such as the kidney, skin, liver, and muscle, enhancing tissue health and restoring the regenerative capacity of aged cells, potentially slowing down age-related decline. Further, by reducing the expression of genes involved in inflammation, senescence, and stress response pathways, partial reprogramming may delay the onset and progression of age-related diseases which contribute significantly to mortality and reduced quality of life in aging individuals.

    Partial reprogramming has thus attracted substantial interest in recent years both from a research and investment standpoint. 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.