AIDS activists in the 1980s
According to the results of my previous post,
some readers reproached me with the fact that I did not describe a plan in it, but only a hypothesis. Although there was one and the other. Just the plan was in general terms, from the height of bird flight. And the hypothesis is very simple: epigenetic coil is epigenetic rejuvenation
. Consequently, by rolling back in any way the
gene methylation profile, we also roll back the biological age of the organism, which means we rejuvenate it.
If someone is interested in a detailed plan of my research, I will present it below. But first I want to answer another objection I heard: “Isn’t it easier to wait for Belmonte?” It’s certainly easier. Sit and wait until others solve all your problems are really easier. But when it comes to fatal illness, this is not the best strategy.
If HIV-infected people in the 80s sat silently and waited, I think a much smaller percentage of them would live to life-saving anti-retroviral cocktails. They understood this very well, and therefore organized many thousands of demonstrations
, demanding to find a cure for HIV as soon as possible, and also created " Dalassian clubs of buyers
" in order to try various experimental therapies on themselves.
I believe that HIV 2.0
should be treated the same way. To do everything possible for the speedy development of at least some effective therapy. Because otherwise, our parents will not live to such therapy.
And I'm not sure that Belmonte is obsessed with this idea as much as I am obsessed with it. But even if he, too, is actively pursuing research in this area, there is nothing wrong with several teams doing this. In any case, Belmonte is primarily a scientist. And I am a practitioner. Only the end result in the form of life-prolonging therapy is important to me, 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 for achieving a much more important goal.
Actually, the research plan
First, a little teaching mother. 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 away too much, you can get cancer, because the cells lose their phenotype and return to the stem, pluripotent state.
Do not forget that it was the ability to effectively return cells to a pluripotent state and served as the main selection criterion for selecting 4 OSKM factors from the original 24 candidates by Yamanaka. Therefore, although the OSKM factors have shown their effectiveness for rejuvenation through partial rollback and constitute a bird in the hand, they are far from ideal for safe rejuvenation. So, it is worthwhile to continue the search for safer ways of epigenetic recoil. It makes sense to start by checking the remaining 20 factors from the original 24 factors of Yamanaki
, and also try to use the Oct4 factor alone, as there is evidence that he alone can roll back epigenetic
and generally is the main “guardian of epigenetic gates."
Also worth checking out are other reprogramming methods developed in recent years. For example, look at the method of replacing the transcription factors Oct4, Sox2, c-Myc with antibodies, which was published literally this week
. If this approach works, then gene manipulations may not be needed at all,
and periodic administration of antibodies will suffice. Although repeated administration may cause problems with a secondary immune response.
In any case, while for me the main hypothesis of therapy is the gene, with the help of certain factors of rollback. But finding the best factors is only half the battle. The other half is how to deliver them 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 showed. Therefore, in order to achieve significant rejuvenation in people, most likely, it will be necessary to activate rollback factors monthly or even weekly.
I see the most economical way to do this is to integrate a special, default inactive gene cassette (containing the 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 with doxycycline; such a cassette with OSKM factors can be delivered to the body using lentiviral carrier available on the market today) and the parallel development of ideal therapy (maximum safe and effective activated by a unique, inert, patentable agent).
Thus, the research can be divided into three parallel tracks:
- develop an optimal dosing regimen using 4 initial factors OSKM
- find safer (not leading to complete de-differentiation) factors or methods of epigenetic recoil
- 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 hand”): develop an optimal dosing regimen using 4 initial factors OSKM
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 OSKM cassette allele is important, since teratomas were formed in mice with 2 copies) OSKM (using doxycycline) inducted 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 decline already from the 2nd day of induction:
This suggests that two-day sequential induction is not optimal. Moreover, the 5-day break seems too long, 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 a 2- or 3-day break).
To test these assumptions, the following steps are necessary (these studies are best ordered from large CROs, for example, from Charles River):
- Buy in Jackson Laboratories wild-type mice with the gene cassette OSKM , preferably at the age of 12 months.
- An 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 in order to track 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 in order to test the hypothesis that mice in the treatment groups have a lower biological age, and to establish the best dosing regimen, not waiting for full mortality in all groups (since only control mice live for 2.5 years, and with the success of therapy, this figure may increase significantly for experimental groups)
- For a GMO pet project, derive several transgenic dog and cat breeds with the OSKM cassette and test various induction modes of these genes, collecting the same data as in mice
Research Track 2 (“cranes in the sky”): search for safer factors of epigenetic recoil (not leading to complete de-differentiation)
- Starting with studies of only Oct4 factor in vitro measuring epigenetic changes (using the Hannum / Horvath methylation clock) - since there is evidence that only this transcription factor is enough for epigenetic rejuvenation
- Ideally, examine each of the 24 original Yamanaki source factors in vitro by measuring epigenetic changes (using the same Hannum / Horvath methylation clock)
- Then examine each combination of identified factors in rapidly aging LAKI mice by creating a new transgenic strain for each combination using a doxycycline cassette.
- Examine the safety of these factors using consistent daily activation (as in Belmonte’s work with OSKM). The goal is to find a better survival rate than showed OSKM factors (from which mice began to die after 3 consecutive days of induction)
- Further, for the safest factors, test new dosing regimens on LAKI mice in order to prolong their lives by more than 50%
- Parallel project: use antibodies instead of factors Oct4, Sox2, c-Myc as in this work . If this approach works, then gene manipulations will not be needed at all . It will be enough to introduce antibodies. Start with LAKI mice, check on WT mice with an eye to changes in methylation hours (will bio-age decrease no worse than from OSKM?)
- Parallel project: explore the role of Piwi proteins and Piwi-interacting RNAs as factors in suppressing transposons and potential epigenetic rejuvenation, as in this article .
- A parallel project: explore the variants of the histones TH2A and TH2B, which, according to this work , can replace Sox2 and c-Myc, which means they themselves can induce an epigenetic regression.
Research track 3: delivery vehicle (carrier) and patentability
- AAV and lentiviruses with 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 efficiency of the delivery of these genes into an adult organism, and (b) the effect on methylation hours and biological parameters from their induction. Will these mice live longer, even if the OSKM genes fall far from all cells? You will need to order and such research.
- For integrative (that is, with the integration of new genes in DNA) approaches, the main obstacle for use in adult animals is low integration (10–50% for AAV or lentiviral vectors), as well as low permeability of the blood-brain barrier (and we, most likely, critically It is important to rejuvenate the hypothalamus). Thus, this should be one of the main areas of research. It is necessary to test sequential multiple transfection - is it possible in this way to achieve the delivery of the desired genes in at least 75% of the cells in all tissues? We need an algorithm for protection against repeated induction in the same cell - if several cassettes with genes fall into it, we do not want them all to be induced.
- Other delivery methods: delivery of proteins or mRNA (non-integrative, but expensive). Order research from companies involved in the development of gene delivery methods. For example, Scarab Genomics .
- Another method: an antibody-released DNA release (new, experimental, expensive method). You will need to consult with its developers.
- To maximize the chances of interest in this therapy to Big Pharma - without which it would be impossible to conduct clinical studies worth hundreds of millions of dollars - one should 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 (as opposed to doxycycline). The cassette itself (shell + inductor) can be developed both on its own and purchased (licensed) as soon as such delivery systems become available.
Here's what I got at prices and terms:
- Research Track 1 (OSKM factors in mice): 1.5–2 million US dollars; term: 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 system): 2-3 million US dollars; term: 2–3 years
- Security: 3-5 million US dollars; term: 2–3 years
- Primate study (optional): US $ 2–2.5 million; term: 1–2 years
- Salaries / Overheads / Patent / Legal and other expenses: 1.5–2 million US dollars per year
Major mileston project:
- 2 years before the introduction to the market of GMOs of domestic animals with OSKM genes induced by doxycycline
- 3-4 years to minimal therapy for adult pets (lentivirus with OSKM or other factors)
- 5–6 years before the start of minimal human therapy trials (IND application to the FDA)
The project duration of 5–6 years means overhead costs in the amount 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 up to 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 arms and legs, which will then begin clinical trials of such therapy (first from atherosclerosis, Alzheimer's disease, diabetes, or other age-related nosologies for which aging drugs have to be tested today) as aging itself is not yet classified by WHO as a disease).
Such is the project of the Moscow Buyers Club crystallized in my head. Or even a “club of self-rescuers”. Let's see what happens. If we manage to find financing for him, I think it will be a glorious hunt. I hope that, as a result of it, HIV 2.0 will suffer significant losses, and we will be able to win at least several decades of healthy life for us and our loved ones.