The work of the drowning themselves

Moscow Buyers Club of aging Therapy – research plan

Yuri Deigin, Geektimes

AIDS activists in the 1980s

Following the results of my previous post, some readers reproached me for describing in it not a plan, but only a hypothesis. Although there was both. It was just a general plan, from a bird's-eye view. And the hypothesis is very simple: epigenetic rollback is epigenetic rejuvenation. Therefore, by rolling back the profile of gene methylation in any way, we roll back the biological age of the organism, which means we rejuvenate it.

If someone is interested in a detailed plan of my proposed research, I will outline it below. But first I want to answer another objection I heard: "Wouldn't it be easier to wait for Belmonte?" Sitting and waiting for others to solve all your problems is really easier. But when it comes to a deadly disease, this is not the best strategy. 

If HIV-infected people in the 80s had sat silently and waited, I think a much smaller percentage of them would have lived to see life-saving antiretroviral cocktails. They understood this perfectly well, and therefore organized thousands of demonstrations demanding to find a cure for HIV as soon as possible, and also created "Dallas Buyers Clubs" in order to try various experimental therapies on themselves.

I believe that HIV 2.0 should be treated in the same way. Do everything possible to develop at least some kind of effective therapy as soon as possible. Because otherwise our parents won't live to see such therapy. 

And I'm not sure that Belmonte is as obsessed with this idea as I am. But even if he is also actively continuing research in this area, there is nothing wrong with having several teams doing this. In any case, Belmonte is primarily a scientist. And I'm a practitioner. I only care about the end result in the form of life-prolonging therapy, and even its commercial value is secondary to me. Although if everything works out, I am sure that commercial success will also come. But money is not an end in itself, it is only a tool to achieve a much more important goal. 

Actually, the research plan

First, a little mother teaching. So, the key hypothesis: in order to reliably rejuvenate the entire body, we need to roll back the epigenetic clock of most cells of the body, if not every cell at all. Thanks to the work of the Belmonte group, we know that this is possible by delivering OSKM factors (or other transcription factors) into the cell. At the same time, the process is subject to the "Goldilocks problem": rolling back too weakly, we will not get a significant rejuvenating effect; rolling back too much, you can get cancer, because the cells will lose their phenotype and return to the stem, pluripotent state.

Do not forget that it was the ability to effectively return cells back to a pluripotent state that served as the main selection criterion for Yamanaka's selection of 4 OSKM factors from the original 24 candidates. Therefore, although OSKM factors have shown their effectiveness for rejuvenation by partial rollback and represent a "tit in the hand", they are far from ideal for the purposes of safe rejuvenation. So, it is worth continuing the search for safer ways of epigenetic rollback. It makes sense to start by checking the remaining 20 factors from the original 24 Yamanaki factors, and also try to use only the Oct4 factor, since there is evidence that he is solely capable of rolling back epigenetics and is generally the main "guardian of the epigenetic gate". 

It is also worth checking out other reprogramming methods developed in recent years. For example, look at the method of replacing transcription factors Oct4, Sox2, c-Myc with antibodies, which was published just this week. If this approach works, then gene manipulation may not be necessary at all, but periodic administration of antibodies will suffice. Although with repeated administration, problems with a secondary immune response may occur. 

In any case, so far for me the main hypothesis of therapy is gene therapy, with the help of certain rollback factors. But finding the best factors is only half the battle. The other half is how to deliver them to the body safely and, ideally, cheaply. The epigenetic aging program is quite stubborn even in the face of weekly kickbacks, as the work of the Belmonte group has shown. Therefore, in order to achieve significant rejuvenation in people, most likely, you will have to activate the rollback factors monthly or even weekly.

The most economical way to do this seems to me to be the integration of a special, by default inactive gene cassette (containing genes for rollback factors) into almost every cell of the patient, presumably using lentiviruses or another integrative delivery method. Further, such a cassette will have to be periodically activated by a unique and inert agent that can be developed separately and will make such therapy patentable. Today, such cassettes are activated, for example, by tetracycline or doxycycline. With this approach, the cost of weekly induction of anti–aging factors will be determined only by the cost of the induction agent (presumably, a small molecule or peptide) - that is, it will be relatively cheap.

The optimal plan seems to me to be a step-by-step, iterative improvement of the already proven approach (induction of OSKM factors using doxycycline; such a cassette with OSKM factors can be delivered to the body using a lentiviral carrier available on the market today) and parallel development of ideal therapy (the safest and most effective factors activated by a unique, inert, patentable agent).

Thus, the research can be divided into three parallel tracks:

• to develop an optimal dosing regimen using 4 initial OSKM factors;
• to find safer (not leading to complete de-differentiation) factors or methods of epigenetic rollback;
• to create the best means of gene delivery (preferably patentable).

Next, I will describe in more detail all three areas of research.

Research track 1 ("tit in the hand"): to develop an optimal dosing regimen using 4 initial OSKM factors

The Belmonte group found that mice begin to die after 3 consecutive days of OSKM induction:

Therefore, they used the 2/5 protocol, in which mice (with only the 1st copy of the allele with the OSKM cassette - this is important, since mice with 2 copies formed teratomas) were subjected to OSKM induction (using doxycycline) for 2 days in a row, and then received a 5–day break before repeating the treatment cycle.

However, the weight of the mice began to decrease from the 2nd day of induction:

This suggests that two-day sequential induction is not optimal. Moreover, a 5-day break seems too long to me, based on what I know about cell cycles. I think the 1/2 or 1/3 treatment regimen has the best long-term potential (1-day induction, and 2- or 3-day break).

To test these assumptions, the following steps are necessary (these studies are best ordered from large CRO, for example, from Charles River):

• Buy wild-type mice with OSKM gene cassette at Jackson Laboratories, preferably at the age of 12 months.
• The ideal would be 30 control mice + 20 groups of 12 mice each for different doses/regimens.
• Run all groups in parallel.
• Periodically collect blood, saliva and urine from some mice from each group to monitor changes in the methylation profile, especially before and after the induction of OSKM factors – to make sure that epigenetic rejuvenation really occurs.
• Some groups should be subjected to cognitive and physical tests to test the hypothesis that mice in the therapy groups have a lower biological age, and to establish the best dosage regimen without waiting for complete mortality in all groups (since only control mice live 2.5 years, and with the success of therapy, this indicator may significantly increase for experimental groups).
• For a project on GMO pets, to breed several transgenic breeds of dogs and cats with an OSKM cassette and test various modes of induction of these genes, collecting the same data as in mice.

Research track 2 ("Cranes in the sky"): search for safer factors of epigenetic rollback (not leading to complete de-differentiation)

• To begin with studies of only the Oct4 factor in vitro, measuring epigenetic changes (using the Hannum/Horvath methylation clock) – since there is evidence that only this transcription factor is sufficient for epigenetic rejuvenation.
• Ideally, examine each of the 24 initial Yamanaki factors in vitro by measuring epigenetic changes (using the same Hannum/Horvath methylation clock).
• Then study each combination of identified factors in rapidly aging LAKI mice by creating a new transgenic strain for each combination using a doxycycline cassette.
• To study the safety of these factors using sequential daily activation (as in Belmonte's work with OSKM). The goal was to find a better survival rate than the OSKM factors showed (from which mice began to die after 3 consecutive days of induction).
• Next, for the safest factors, test new dosing regimens on LAKI mice in order to prolong their life by more than 50%.
• Parallel project: to use antibodies instead of factors Oct4, Sox2, c-Myc as in this work. If this approach works, then gene manipulation will not be needed at all. The introduction of antibodies will be enough. Start with LAKI mice, and check on WT mice with an eye to changes in the methylation clock (will the bio-growth decrease no worse than from OSKM?).
• Parallel project: to investigate the role of Piwi and Piwi-interacting RNAs proteins as factors of transposon suppression and potential epigenetic rejuvenation, as in this article.
• Parallel project: to investigate variants of histones TH2A and TH2B, which, according to this work, can replace Sox2 and c-Myc, which means they themselves can induce epigenetic rollback.

Research Track 3: Delivery vehicle (carrier) and patentability

• AAV and lentiviruses with the OSKM cassette can be purchased now. It is necessary to take LAKI and WT adult mice and conduct a series of experiments on (a) the effectiveness of delivering these genes to an adult organism, and (b) the effect on the methylation clock and biological parameters from their induction. Will such mice live longer, even if OSKM genes do not get into all cells? It will be necessary to order such studies.
• For integrative (that is, with the integration of new genes into DNA) approaches, the main obstacle to use in adult animals is a low degree of integration (10-50% for AAV or lentiviral vectors), as well as low permeability of the blood-brain barrier (and it is most likely critically important for us to rejuvenate the hypothalamus). So this should be one of the main areas of research. It is necessary to test sequential multiple transfection – is it possible to achieve the delivery of the necessary genes in at least 75% of cells in all tissues in this way? We need an algorithm to protect against multiple induction in the same cell – if several gene cassettes get into it, we don't want them all to be induced at all.
• Other shipping methods: delivery of proteins or mRNA (non-integrative, but expensive). Order research from companies involved in the development of gene delivery methods. For example, at Scarab Genomics.
• Another method: antibody-released DNA release (a new, experimental, expensive method). It will be necessary to consult with its developers.
• In order to maximize the chances of a Large Pharma being interested in this therapy, without which it will be impossible to conduct clinical trials worth hundreds of millions of dollars, it is necessary to strive not only for a safe and effective, but also for a patentable approach. Ideally, it should be a cassette with a unique combination of factors activated by a safe, inert and patented activator (unlike doxycycline). The cassette itself (shell + inductor) can be developed either on its own or purchased (licensed) as soon as such delivery systems become available.

Prices, terms

Here's what I got for prices and terms:

• Research track 1 (OSKM factors in mice): 1.5–2 million US dollars; duration: 3 years.
• Research Track 1 (pets): 3-4 million US dollars; term: 2 years.
• Research track 2 (best factors): 6-8 million US dollars; term: 4-5 years.
• Research Track 3 (delivery vehicle): 2-3 million US dollars; term: 2-3 years.
• Security: 3-5 million US dollars; term: 2-3 years.
• Primate study (optional): 2-2.5 million US dollars; duration: 1-2 years.
• Salaries / Overhead costs / Patent/Legal and other expenses: 1.5–2 million US dollars per year.

The main milestones of the project: 

• 2 years before the market launch of GMO pets with OSKM genes induced by doxycycline
• 3-4 years to minimum therapy for adult pets (lentivirus with OSKM or other factors)
• 5-6 years before the start of trials of minimal therapy in humans (IND application stage to the FDA)

The project term of 5-6 years means overhead costs of 7.5–12 million US dollars, based on the above 1.5–2 million US dollars per year. Thus, the total amount of expenses before the IND stage is: 24-37.5 million US dollars.

If the project successfully reaches the IND stage, then Big Pharma companies will tear it off with their hands and feet, which will then begin clinical studies of such therapy (first from atherosclerosis, Alzheimer's disease, diabetes or other age-dependent nosologies, according to which today it is necessary to test drugs to slow aging, since aging itself has not yet been classified by WHO as a disease).

Resume

Such is the project of the "Moscow Buyers Club" crystallized in my head. Or even the "club of self-rescuers". Let's see what happens. If we can find funding for it, I think it will be a nice hunt. I hope that according to its results, HIV 2.0 will suffer significant losses, and we will be able to win back at least several decades of healthy life for us and our loved ones.

Portal "Eternal youth" http://vechnayamolodost.ru  19.09.2017