March 12th, 2023
Here's update #2 on LEVF's "round 1" attempt to achieve robust mouse rejuvenation!
Yesterday we started the treatment of cohort 2. As I explained two weeks ago, this cohort consists of 300 mice, rather than the 200 that we have in cohort 1. The three 100-mouse treatment groups that have just begun their journey are numbers 3, 5 and 8 in the project description, respectively receiving:
only senolytic
only haematopoietic stem cells (HSCs)
rapamycin, senolytic and HSCs
There's plenty to say about each of these, so let me dive in.
First the senolytic. We cogitated for a long time about what senolytic to use, and you may recall that we didn't even specify it in the initial description of the project - but eventually, as I've mentioned in a few recent interviews, we decided on galactose-conjugated Navitoclax. Navitoclax has a reasonable pedigree as a senolytic, but it's quite toxic to platelets, so several years ago Manuel Serrano had the brilliant idea of improving its specificity for senescent cells by encasing it in galactose. His logic was that galactose will be broken down (and the drug thus liberated) preferentially in senescent cells, on account of their (non-universally, but typically) high level of expression of lysosomal beta-galactosidase.
It worked pretty well, and in more recent work Manuel and his colleagues made a further improvement by, rather than encasing the drug, covalently conjugating it to a single galactose molecule to create an inactive, but galactosidase-activatable, "prodrug". We worked with a top chemistry CRO in Europe to synthesise this prodrug at the scale we needed. We are replicating the treatment regimen used in prior work, which entails injecting the stuff every day for ten days. In due course we will make a decision on whether to repeat the protocol, based on data as to the rate of re-emergence of senescent cells, but that won't be for at least six months.
So to the HSCs. One of the most high-profile lines of research in the biology of aging over the past decade or two has been the rejuvenating impact on old animals of blood from young animals. More recently, this work has mostly focused on the contribution of the cell-free component of the blood, i.e. plasma. (I won't opine here on the debate as to whether it's good stuff in young plasma or bad stuff in old plasma that matters more.) But it also turns out that cells from young blood matter, as shown by a couple of studies that transplanted HSCs from young mice to old ones and obtained very good life extension. So we are doing that.
The process is quite involved, consisting of two separate steps: first recipients undergo a 5-day preconditioning regimen, using G-CSF and Mozobil (AMD3100) to mobilize old HSCs into the blood; then, at the end of the 5th day, mice receive HSCs from young donors. I should also explain what I mean by "HSCs": we isolate cells using a standard procedure termed lineage-depletion. Some groups go further and specifically isolate "long-term repopulating" HSCs, but the above studies didn't, so we chose not to do so either.
Finally there's the first multi-treatment group, consisting of rapamycin, senolytic and HSCs. There's nothing different here about the specifics of administration, but I do want to mention one thing: we are not administering the injected treatments all on the same day(s). There are various reasons for this. The first is that injections are rather stressful to the mice, and based on decades of experience it has become standard practice to do at most two injections per day; since the mobilisation protocol involves twice-daily G-CSF, that means the senolytic needs to be before or after. We've chosen to do the HSCs first, followed by the senolytic; there were arguments both ways, but we decided that the main consideration was the possibility that mobilisation would have some similarities to a wound, with the implication that having a bit of SASP around might facilitate re-engraftment.
In that connection I may as well mention telomerase, even though that isn't coming until cohort 3 two weeks from now. We've decided to do the telomerase gene therapy last of the three injected treatments, after both rejuvenation of the stem cell niche and ablation of senescent cells, in order to avoid inadvertently "de-senescing” cells that became senescent for a beneficial reason, such as to stop themselves becoming cancerous.
I'll also mention that this sequencing of interventions has been very much ongoing for the past two weeks, in cohort 1, even though none of those 200 mice were/are receiving any of the injected interventions. That's because of our choice to evaluate the impact of the method of delivery of an intervention, independently of the intervention itself, by doing two types of control, mock and naive. See my post of two weeks ago for more details, but the upshot is that half the mice in each of groups 1 and 2 (group 2 is getting rapamycin, remember) are getting mobiliser without HSCs, senolytic vehicle without senolytic, and empty (i.e. lacking the telomerase gene) virus, while the other half are getting none of those.
Now for the final topic: deaths. I've decided to be quite expansive here, because we've committed to giving very frequent updates on this, but those of you who are not utterly immersed in the project (which means all of you!) are at risk of overinterpreting the very early data. So I want to go into some depth, concerning not only how things are going but also how we are monitoring the data.
First the bald facts. As of now, two weeks in for the first 200 mice, we have had four deaths. The first was 11 days ago, so only three days into the experiment, and it was an "all controls" animal, which had received only a few doses of G-CSF. The other three, of which one was getting rapamycin and the other two were getting all controls, died early this week.
Now for how we are thinking about these deaths. The overall statistical significance of the above numbers is of course negligible. However, at this initial stage we are being absolutely eagle-eyed concerning any possibility that one or other of our treatments might be doing harm to our animals. That possibility divides into two options: that the treatment itself is toxic, or that the mode of delivery is toxic. (See above!) In the latter case, it is not wrong to respond by adjusting the mode of delivery, if an alternative is available. So, early this past week we looked very closely at the three more recent deaths - and we were rather worried; we noticed that all of them were in the mock-control group (for everything injected; as noted, they were split as regards rapamycin), not the naive group. Unlike the mouse that died 11 days ago, they had just started receiving the senolytic vehicle (which is a cocktail of solvents, because Navitoclax is quite hydrophobic), having completed their course of G-CSF and Mozobil.
These coincidences must be evaluated at once in order to decide what to do. But the thing I must massively stress is that any such evaluation must consider how many alternative ways in which a similarly conspicuous coincidence could have occurred, in order to decide whether it is suspicious enough to act upon. Our option, in this case, is to retreat to a simpler vehicle, pure PBS, even though Navitoclax may not dissolve so well. I decided that we should delay doing that, pending further data; if we saw no more deaths fitting the pattern of those three, let's not change anything, but if we see a few more, let's go to PBS, with the adjustment of statistics that that implies. Fortunately, there were no more deaths in the next three days, so controls continue to receive the vehicle formulation as planned.
As the experiment proceeds, these judgement calls will continue to need to be made, but they will become progressively easier because they'll be based on more data. But I wanted to fill you in on the above - partly for full disclosure, but mostly to let you know how much care we’re taking here. We are conducting the most ambitious mouse lifespan experiment of all time, and we're not doing it blindly.