Robust Mouse Rejuvenation Study Started

Aubrey De Grey gave a kickoff announcement for the new Robust Mouse Rejuvenation study.

Above- Just a random picture of mice, not the mice in this study. The study just has a few hundred ordinary mice that have not had anything special so far.

The Robust Mouse Rejuvenation” study that LEVF (Longevity Escape Velocity Foundation) is funding at Ichor Life Sciences in Syracuse.

Donate to the Longevity Escape Velocity Foundation at this link.

They planned this immensely complex study in such a way that they could begin it as soon as all the prerequisites (including masses of custom reagents) were in hand, subject to an age at onset no younger than 18 months, but knowing that any age up to 20 months would be fine. In the end they are starting at 19 months, which for the 200 oldest mice was a few days ago. You may wonder whether mice have been dying before the experiment begins: the answer is, yes a few, but only VERY few. There are enough extras to be able to compensate.

The ultimate goal in this program is to achieve “Robust Mouse Rejuvenation”. They define this as an intervention, almost certainly multi-component, that:

is applied to mice of a strain with a historic mean lifespan of at least 30 months

is initiated at an age of at least 18 months

increases both mean and maximum lifespan by at least 12 months

In each study in this program, they will examine the synergy of (typically at least four) interventions already known individually to extend mouse lifespan when started in mid-life.

A Lot of Mice

They bought mice in four groups (cohorts), each with a birthday range of only a day or two, but with groups spaced by intervals of two weeks. They also wanted to have each of the ten treatment groups be entirely within one cohort, so these ended up being split not as four groups of 250 mice but as 200, 300, 200 and 300. So the first 200 mice are 19 months old today (+/- a day or so), the next 300 will reach 19 months of age on March 11th, the next 200 on March 25th, and the last 300 on April 8th.

The first cohort, whose treatment starts today, will have relatively simple treatment regimens. These 200 mice will constitute treatment groups 1 and 2, which means that half of them, i.e. 50 males and 50 females, will comprise the “all controls” group, and the other half the “only rapamycin” group. Both of these groups will actually be further split in two, with 25 of each sex receiving “mock” control treatments and the other 25 receiving “naive” controls. What distinguishes the two? Well, “mock” means they do everything to the mice that they would if they were getting the intervention, except that they don’t actually give the intervention itself, whereas “naive” means they do nothing. The purpose of this is to be able to factor out any impact on lifespan or healthspan that the delivery mechanism may have. So, for example: they are administering rapamycin in the mice’s food, but they are doing it in a particular way that has worked well for other groups, namely encased in microcapsules. So, half of the mice that are NOT getting rapamycin will just get regular food, but the other half will get food containing empty microcapsules. Similarly, the telomerase gene therapy will be delivered virally, so among the mice that are not getting telomerase (which includes all 200 of the mice whose journey begins today), half will receive no virus, and half will get virus without telomerase.

Cohort 2 will be the first multi-intervention group. This will indeed be another level up from what Aubrey outlined for Cohort 1.

This study is costing about $3M.

Longitudinal Assessment: All living animals will undergo functional and behavioral assessment at timepoints linked to % group survival (see Endpoints below). These assessments have been selected on the basis of being minimally invasive and stressful, while simultaneously allowing evaluation of parameters reflecting functional capacity in mice.

They will include rotarod and grip strength assessments for motor and muscle function, novel object recognition for cognition, and software-enhanced open field testing to measure movement velocity and duration, gait abnormalities, etc. as well as provide indications of openness vs anxiety. Frailty will also be scored on the basis of body condition, weight, and factors such as degree of kyphosis and alopecia on an ongoing basis.

In addition, blood samples will be taken longitudinally from all living mice for glucose tolerance testing, platelet counts, and to screen for HSCT engraftment (see Interventions). A fraction of each sample will be banked for retrospective analysis of the longest-lived mice.

Endpoints: Male and female mice of each intervention group will be sacrificed on a declining schedule based on in-group survival (see culling schedule). An equal number of animals will be randomly selected from those receiving mock or absent control interventions (if any) for a given intervention group.

At these timepoints necropsy will be performed, along with CBC with leukocytes (to assess immune status), blood chemistry, and storage of all major organs and tissues. Downstream analyses will include measurements of serum cytokines/chemokines (inflammatory state), telomere length, and epigenetic age estimation, in addition to tissue histology.

The same intensive post-mortem analysis will also be carried out on every 4th mouse that is euthanized for humane reasons, while tissue and biofluid storage will be conducted on all expired animals where the time of death permits the collection of valid samples.

Interventions

Rapamycin
Rationale: Rapamycin has been selected as our baseline intervention due to the many studies demonstrating a lifespan effect in mice, including when started late in life. mTOR inhibition by rapamycin is believed to delay the onset of cancer, an effect which may be potentiated by combination with other therapeutics.

Design: Mice in treatment groups will receive 42ppm Eudragit S100 enteric-coated rapamycin in chow (Purina 5LG6), using the same encapsulation provider and formulary as ITP studies. Food will not be irradiated. They have selected oral delivery in chow over other delivery methods in order to be minimally invasive and so that the drug can be administered continuously.

A dose of 42ppm has been chosen on the basis of sex-specific dose effects [PMID 33145977], particularly the observation that male mice receive minimal or no benefit from lower doses of 14ppm [PMID 24341993], and higher doses have not shown any detrimental effects.

Half of the mice NOT receiving rapamycin will receive chow containing empty Eudragit, while the other half will have standard chow.

Hematopoietic Stem Cell Transplant
Rationale: A reduced regenerative capacity of stem cells is widely believed to contribute to age-related morbidity and functional tissue decline. Numerous studies have shown stem cell transplantation to have rejuvenating effects in mouse tissues, and several additionally show a lifespan extension effect [PMID 32012439, 31031800].

Design: The study design is largely (but see below) based on the protocol utilized by Guderyon et al. 2020 [PMID 32012439] and consists of mobilizing the recipient bone marrow niche followed by transplant of lineage-depleted hematopoietic stem cells (HSCT).

Bone Marrow: They have opted to use lineage-depleted bone marrow HSCs as opposed to additional selection for and expansion of long-term self-renewing HSCs. This was on the basis that 1) prior lifespan studies used whole or lineage-depleted bone marrow, which may include MSCs or other beneficial cells promoting engraftment, and 2) expansion protocols have not been extensively validated.

Telomerase Expression
Rationale: Though they recognize that telomerase gene therapy may be neither practical nor especially beneficial for humans in the near term, it has been shown to significantly extend the lifespan of naturally-aged mice without increasing cancer incidence [PMID 22585399], while also improving numerous measures of healthspan. This work, originally conducted using AAV-mTERT gene therapy, has been replicated in numerous genetic and disease models [PMID 31624261, 27252083, 29378675], and recently was adapted as a CMV-based gene therapy [PMID 35537048], enabling repeat dosing in rodents and humans.

Design: The present study attempts to mimic, as closely as possible, the recent work conducted by Church, Parrish et al. [PMID 35537048]. For this, they will utilize a CMV-mTERT gene expression vector, administered monthly to mice from the age of 18 months. As in the above, they will utilize intranasal delivery, which the authors demonstrated to be equally beneficial versus IV administration and which allows us to limit unnecessarily invasive procedures.

Furthermore, the model study stopped treatments upon death of all the control mice ~29 months, then resumed treatments at 32 months given the excellent health demonstrated by treatment groups. Despite this pause, aged mice that resumed mTERT therapy lived to an average 37.5 months, a 41.4% increase over controls. They have therefore chosen to continue mTERT therapy on a monthly schedule throughout the remaining lifespan of the intervention groups.

Senescent Cell Ablation
Rationale: Senescent cells (SnC) are shown to accumulate with age in nearly all tissues, and multiple studies have now shown improvement in healthspan parameters upon SnC removal. Although few of these studies emphasize lifespan effects, we hypothesize that SnC removal is important for mouse longevity due to the role played in immune decline and in cancer – the two leading causes of death in C57Bl/6J mice. They believe an effective senolytic may not only reduce cancer incidence by removal of cells which are cancer-capable (via senescence escape), but also by improving local immune surveillance against abnormal cells, by reducing the SASP’s cloaking effect and relieving systemic immune fatigue from SASP-driven chronic inflammation. In this way, it is also possible that SnC removal can reduce the age-related increase in susceptibility to pathogens.

Design: They are currently in advanced discussions with several groups concerning the choice of senolytic and its delivery method. They will update this section as soon as the final decision is made on these matters.

Future Interventions
Though they plan to conduct subsequent iterative studies based on insights from this first study and from ongoing discoveries by others, they also have several high-priority interventions which we were not able to include in the current study due to manufacturing timelines or cost.

One such intervention is apheresis or plasma dilution, which have each now demonstrated rejuvenating effects in multiple tissues in both mice [PMID 32474458] and humans [PMID 35999337], probably by diluting inflammatory molecules and damaged proteins in the circulation. Another alternative may be direct infusion of recombinant albumin, which has shown promising preliminary lifespan results in a preclinical model [PMID 34439857].

They are also enthusiastic about next-generation senolytic technologies, and are eager to include tailored approaches such as transcription-based biosensors under genetic control, tissue-specific senolytic prodrugs, and PROTAC-senolytics in subsequent intervention trials.

Laboratory mice also often succumb to common infections due to weakening of the immune system. T-cell rejuvenation is an aspect not specifically addressed in the present study, and may serve to support robust immune responses to pathogens thus preventing premature death from infection. Cellular reprogramming is one method being investigated to accomplish rejuvenation of the immune system.