Another great comment from Michael Rae on why CRISPR-Cas9 is not ready for in vivo gene editing

https://www.fightaging.org/archives/2016/04/the-actuarial-press-interviews-aubrey-de-grey/#comment-23917

The part relevant to using CRISPR-Cas9 for in vivo gene editing:

“But your (Barbara’s) implicit premise seems to be that the CRISPR/Cas9 system could easily be used for somatic gene therapy — ie, introducing therapeutic genes into existing tissue in situ. And if you didn’t mean that, such is certainly the explicit premise of others in the comments to this thread, and in previous comments in FA! to which I’ve not previously responded. In fact, using the CRISPR/Cas9 system for this purpose would require a great deal of innovation, including the emergence of strategies that no one has yet identified, even in principle.

For one thing, CRISPR/Cas9 does not come with any kind of vector system to deliver both components of it, and we don’t have a sufficiently safe and effective delivery vector for it, although people are certainly working on delivery systems all the time. And whereas most delivery systems for gene therapy currently in use are for a single active component, it’s actually at the moment very difficult even in cell culture to assure the coincident presence (and expression, as relevant), in the same cell at once, of all of the components of the system (the Cas9 exonuclease, the guide RNA, and the exogenous repair template) — let alone to do this in vivo across an entire somatic tissue. Additionally, the payload size that the system can handle is as of yet far too limited to be used for any kind of gene therapy useful to us, although people are working on that, too.

There are much larger problems, however, that are more central to the genome-editing mechanism of the system itself. The system works because the Cas9 endonuclease makes double-strand breaks in the host cell genome (or, using the trickier, engineered “dual nickase” system, you use two modified Cas9s that each make its own single-strand breaks at one end of the desired insertion site) at a site determined by the guide RNA, and the cell’s DNA repair machinery “repairs” the break with new genetic material introduced in a separate repair template. The cell can use one of two DNA repair mechanisms to do this: the Non-Homologous End Joining (NHEJ) pathway or the Homology Directed Repair (HDR) pathway.

NHEJ is more or less OK for making decent-sized loss-of-function mutations (which scientists often want to do), but is too sloppy to be used for insertion of therapeutic genes, whether in mice or in humans: it often leaves small insertions or deletions that bridge over the site of the break in the strand, resulting in either frameshift mutations or premature stop codons within the open reading frame of the gene, resulting in either a protein that is prematurely truncated and useless or producing useless mRNA that just gets degraded by nonsense-mediated decay (or, theoretically, even a gene with a deleterious gain-of-function mutation, though I’ve never heard this raised as a serious prospect).

HDR is very precise, but it has a very low (<10% of modified alleles) efficiency, and it is only active in cells that are actively in the process of dividing, making it useless for introducing therapeutic genes into mature neurons or heart and skeletal muscle cells, and for substantial numbers of cells in organs like the liver and the skin at any given time."

The full comment:

"To start with a direct quote of the passage that has raised most of the discussion:

De Grey readily admits that the likelihood of his research successfully extending his own lifetime is low. "I put it at 20-25 years from now when we have a 50-50 chance of getting to a decisive level of comprehensiveness that works, which I've called longevity escape velocity. If we do get there by then, I've got a fair chance of benefiting. But I have absolutely no doubt there's at least a 10% chance we won't get there for another 100 years because we hit new problems that we haven't thought of. So if I look at my own personal prospects, or the prospects of any other particular person, the timelines and uncertainty result in this all being very speculative."
Posted by: CANanonymity at April 9th, 2016 2:10 PM: "decisive level of comprehensiveness that works," I'm not sure what he means, but I'm guessing 'good enough', 'we understand our stuff enough' that we can make the rest of the therapies from now on.

As he indicates, this refers to the central thesis of actuarial escape velocity, which (as you seem to understand) means that we have achieved the first, decisive, comprehensive panel of rejuvenation biotechnologies: a panel that would, amongst them, effectively remove, repair, replace, or render harmless a sufficiently wide range of forms of cellular and molecular damage, and do so effectively and safely enough, to grant 20-30 years of additional life expectancy to a person in late middle age — which (to address one of your other confusions) is what he means by robust human rejuvenation (RHR).

This would mean that we can make the rest of the therapies from [then] on, in the sense that the quater century of increased life expectancy enjoyed by those first beneficiaries would be sufficient time to develop further refinements and additions to the panel, expanding its efficacy, safety, and comprehensiveness sufficiently well to increase life expectancies by a further 30%, which in turn would give them even more time alive — and, again, more time for medical science to deliver yet more and better rejuvenation biotechnologies. And so on, until we had aging under complete medical control, and life expectancy would be bounded only by causes unrelated to aging (violence, accidents, natural disasters, catastrophic infections, etc).

Posted by: CANanonymity at April 9th, 2016 2:10 PM: In 20-25 years, he and his SENS will not have All the 6 or 7 therapies made. If he implied that, he forgot his own longevity paper where he said the first therapy will take 25 years rough.

No, he did not: he said (as he repeats above) that with sufficient funding, we would have a 50-50 chance of having the first comprehensive panel of rejuvenation biotechnologies sufficient to achieve RHR. Such a panel would require not just one, nor even seven individual rejuvenation biotechnologies (since there are several key aging lesions that will need to be repaired to achieve RHR even within the seven categories of cellular and molecular damage of aging), but a dozen or more.

Posted by: CANanonymity at April 9th, 2016 2:10 PM: And then it will be quicker/easier/more experienced for new ones..so a 6 years wait for each other new therapy. Totaling more than 50 years. I think he meant just for the first one that's it, to have some kind of 'direction/momentum' to create the 6 others.

The paper that you're misremembering models what happens after the first, comprehensive panel of rejuvenation biotechnologies is achieved. The model is premised on further iterations of rejuvenation biotechnologies cutting by half the impact of each category of aging damage on health and functionality every six years. It would then take 42 years to complete such a cycle for all seven categories of aging damage.

At the other end:

CANanonymity at April 10th, 2016 10:49 AM: As Michael Rae said, Reason, me and others, damage must be entirely wiped out to increase maximum lifespan

Here you are falling into a similar error at the other end of the process. If damage must be entirely wiped out to increase maximum lifespan, there would be no point in iterating further improvements on or additions to the first achievement of RHR. Rather, as I said in the post you're misremembering (at February 3rd, 2016 4:46 PM), "To move the needle on maximum lifespan, you have to push back on all of the cellular and molecular damage of aging, not just one form." That is, in order to achieve RHR (and initiate LEV), the panel of rejuvenation biotechnologies must be comprehensive, removing or repairing substantial amounts of all of the categories of aging damage, inclusive of the specific instances within each category that actually limit the current "normal" lifespan.

But you don't have to "wipe out" all such damage to proportionately extend the maximum potential life that a given person can live. Remember, even newborn infants have some level of aging damage in their tissues, but they still have many decades of life ahead of them, because in no tissue is has the burden of aging damage reached a "threshold of pathology" at which diseases of aging emerge and life becomes threatened. Thus, the goal is only to remove or repair sufficient amounts of each cellular or molecular lesion in the tissues to restore tissue integrity to levels equivalent of a person sufficiently younger, who has a given amount of potential lifespan ahead of him or her, to give that same potential to the recipient of the therapies.

The reciprocal of this, of course (and what you had misremembered or misunderstood), is that you can't push back on just one or just a few forms of aging damage and expect an increase in maximum potential lifespan for any individual, because of the "weakest link in the chain" problem: other forms of aging damage will continue to rise to pathological levels on more or less their original trajectory, even as the one(s) that you have reduced are pushed further away from that threshold. There is somewhat of an analogy here to genetic disorders that cause greatly-accelerated accumulation of the remaining forms of aging damage, such as the mutations in APP or PS1 that cause familial, early-onset Alzheimer's disease, or those in Parkin or PINK1 that cause young-onset Parkinson's (but in all of the remaining forms of damage instead of just one): the person is young in many respects, but on a severe trajectory toward morbidity and mortality because of a high burden of a subset of "normal" aging damage.

The goal, brief, requires comprehensive reductions in the burden of aging damage, leaving a person with the structural integrity of a much chronologically younger person who still has many decades ahead of him or her — not that the burden of all aging damage has to be "wiped out" (brought down to zero).

Posted by: CANanonymity at April 10th, 2016 10:49 AM: The question that lingers, is what will RHR SENS do to already aged people who are 100, 110 super centenarians ? Are they game over too late ? We talked about this, damage is too high in them for salvaging, despite RHR. Aubrey de Grey said we have to do this in middle-life (his age, 53 years) to gain from RHR. RHR in 25 years, (for the 25 years old of today), will gave them that chance. Will a 75 year old AdG gain from RHR to get to LEV ? If AdG means that RHR is a 30 year health extension (that can be repeated ??), I don't see how RHR can make AdG go above 122 ever because he implies that RHR is using all 7 therapies or many of them; how then can this repair *all* damages and make any human overcome MLSP and reach LEV. I don't question to the greatness, it is a great thing, I just am curious (and worried) that it flops.

You're certainly right that as a person suffers with increasing burdens of aging damage, our ability to rejuvenate them effectively and safely declines, eventually reaching a null or negative point, and that this plays into Dr. de Grey's assessments of his own odds of reaping the full benefits of rejuvenation therapies himself. This is itself modeled in the paper you've misrecalled or misunderstood (Figure 5), so this is not a question that still "lingers" in the sense that no one has thought of or addressed it, or to which no one has an answer beyond idle speculation. When inspecting the graph, note again that the ages given are ages for receiving the first comprehensive panel of rejuvenation biotechnologies with ongoing iterative improvements, not the age that the first individual "damage-repair" therapeutic arrives; and that the y-axis is the proportion of the original cohort of a given age at receipt of first full treatment still alive by a specific age, so that once a given survival trajectory reaches its plateau, all or very nearly all of the remaining members of the original cohort survive until killed by something other than aging.

Posted by: CANanonymity at April 9th, 2016 2:10 PM: "…which I've called longevity escape velocity." It's good he remembers (of course : D…he wrote it), but he is standing by his research paper. Although, I clearly 'Sens'e (pun intended, had to : D, that he is trying to not make extreme outlandish claims, because he knows his longevity escape velocity paper would allow immortality by rejuvenation; which does not sit well with people (like the fatalists) who don't believe in rejuvenation, as we wish, that could allow eternal life. And he knows that everyone knows his SENS therapies could allow that, so he doesn't want to give false hopes if it fails to even rejuvenation all that much.

Dr. de Grey has never, ever backed away from the actuarial implications of the "damage-repair" heuristic of SENS: such is, indeed, the whole basis of the talk he gave that sparked this discussion. On the other hand, neither the escape velocity paper, nor the mathematical model of the progress of rejuvenation biotechnologies, nor anything else he has said asserts or implies that rejuvenation biotechnology will allow immortality by rejuvenation or allow eternal life. It would "only" dramatically extend life expectancy by eliminating age-related causes of ill-health and death.

MissKaioshin at April 9th, 2016 4:54 PM: This is significant because Aubrey de Grey himself is admitting that our chances are extremely slim. I've said before that none of us are going see longevity escape velocity in our lifetimes,

It is a mischaracterization of Dr. de Grey's views to say that he "is admitting that our chances are extremely slim." The author of the press story summarizes Dr. de Grey's assessment of his own prospects as given above based on the timeline that the journalist translates as indicating an "extremely slim" chance for Dr. de Grey. Whether "a 50-50 chance" of achieving robust human rejuvenation within 25 years, intersecting with the current lack of the funding, or the trajectories laid out in the paper and results graph discussed above, should be characterized as "extremely slim" chances for Dr. de Grey or for any individual is a qualitative and necessarily somewhat subjective judgement.

To be meaningful, such characterization needs to be informed by a given person's current age and health and risk factors, and will ultimately depend on how quickly we will collectively succeed — through philanthropic giving, advocacy, and political action — in initiating the necessary radical reorientation of global biomedical research: away from the current approach of modulating aberrancies of metabolism and "managging" metabolic risk factors, and toward the "damage-repair" model necessary for the comprehensive prevention and reversal of the diseases of aging. Simply on their face, the journalist's gloss is on the pessimistic side, though perhaps not as outrageous as Barbara T or Slicer's initial take on the matter.

Posted by: Barbara T. at April 9th, 2016 6:02 PM: This [timescale] flies in the face of a number of very important scientific discoveries … as well as the existence of therapies for which there is proof of concept (e.g. senescent cell and transthyretin amyloid clearing) and that are already on the menu of various start-ups.

… and similar comments by John Anderson. This is all very good work; I might also highlight the even more advanced state of development of immunotherapies targeting the removal of beta-amyloid, alpha-synuclein, and malformed tau, all of which are now in human clinical trials, as well as ongoing support for cell therapy. However, we have to remember the "weakest link in the chain" problem, as discussed with regard to the similar case of clearance of large numbers of senescent cells: to achieve RHR will require successful targeting of each and every one of the various cellular and molecular aging lesions that currently contribute significantly to limiting human life expectancy due to aging. There is currently good investment in a minority of areas of rejuvenation research, and happily a few startups going after some of the less well-supported ones, but even those lean more toward targets that are perceived to be low-hanging fruit: one high priority for SENS Research Foundation is therefore to use critical-path analysis to invest our very limited resources in areas that are as yet being neglected by such players.

I would note that you list amongst your examples ongoing progress on true clearance of wild-type transthyretin amyloid. This is itself work funded by the Foundation.

Posted by: Barbara T. at April 9th, 2016 6:02 PM and 7:11 PM: This [timescale] flies in the face of a number of very important scientific discoveries such as CRISPR Cas9 (in 2003 de Grey himself said that the fact that gene therapy was "still rather black magic" was a major obstacle to the implementation of SENS) … [T]he funds used to develop CRISPR Cas9 cannot be considered SENS-related and yet the technique advanced – or is going to – the cause greatly.

I think you're thinking about this somewhat backwards. The budget was intended to include only core work on rejuvenation biotechnology: it assumed the ongoing progress in life sciences generally, including above all things like gene therapy, which is a robustly-supported area of research whose medical value is very widely accepted in biomedical research and only modestly controversial in public discourse. The original timescales therefore essentially assumed the emergence of robust gene therapy and other tools of widespread biomedical utility out of general biomedical research — and remember that it was made at a time when the NIH budget "doubling" (so-called) was still ongoing and expected to continue, rather than suddenly being flatlined or declining in the face of inflation beginning in the middle of the previous administration. And even very robust gene therapy is an enabling tool for delivering rejuvenation biotechnology, rather than itself one of the key therapies that needs to be developed.

As to the CRISPR/Cas9 system specifically: this is certainly a very useful tool for biomedical research, with utility for delivery of some SENS therapies as well — notably, editing stem cells to enable some therapeutics (and to make them impregnable to cancer), because those can be modified ex vivo, and defective cells can be screened and discarded, while successful transfectants can be expanded and seeded back into the tissue.

This kind of approach can also be used for nearer-term medical applications not directly related to rejuvenation goals as such: for instance, the Regeneron/Intellia agreement highlighted by Jim, involving ex vivo manipulation of haematopoietic cells for CAR-T cell therapy and other haematopoietic applications, as well as for liver cells in people receiving a liver transplant as part of treatment for hereditary transthyretin amyloidosis resulting from an aggregation-prone mutant transthyretin gene (a quite different problem from dealing with senile systemic amyloidosis, where the normal, wild-type protein aggregates due to stochastic damage).

But your (Barbara's) implicit premise seems to be that the CRISPR/Cas9 system could easily be used for somatic gene therapy — ie, introducing therapeutic genes into existing tissue in situ. And if you didn't mean that, such is certainly the explicit premise of others in the comments to this thread, and in previous comments in FA! to which I've not previously responded. In fact, using the CRISPR/Cas9 system for this purpose would require a great deal of innovation, including the emergence of strategies that no one has yet identified, even in principle.

For one thing, CRISPR/Cas9 does not come with any kind of vector system to deliver both components of it, and we don't have a sufficiently safe and effective delivery vector for it, although people are certainly working on delivery systems all the time. And whereas most delivery systems for gene therapy currently in use are for a single active component, it's actually at the moment very difficult even in cell culture to assure the coincident presence (and expression, as relevant), in the same cell at once, of all of the components of the system (the Cas9 exonuclease, the guide RNA, and the exogenous repair template) — let alone to do this in vivo across an entire somatic tissue. Additionally, the payload size that the system can handle is as of yet far too limited to be used for any kind of gene therapy useful to us, although people are working on that, too.

There are much larger problems, however, that are more central to the genome-editing mechanism of the system itself. The system works because the Cas9 endonuclease makes double-strand breaks in the host cell genome (or, using the trickier, engineered "dual nickase" system, you use two modified Cas9s that each make its own single-strand breaks at one end of the desired insertion site) at a site determined by the guide RNA, and the cell's DNA repair machinery "repairs" the break with new genetic material introduced in a separate repair template. The cell can use one of two DNA repair mechanisms to do this: the Non-Homologous End Joining (NHEJ) pathway or the Homology Directed Repair (HDR) pathway.

NHEJ is more or less OK for making decent-sized loss-of-function mutations (which scientists often want to do), but is too sloppy to be used for insertion of therapeutic genes, whether in mice or in humans: it often leaves small insertions or deletions that bridge over the site of the break in the strand, resulting in either frameshift mutations or premature stop codons within the open reading frame of the gene, resulting in either a protein that is prematurely truncated and useless or producing useless mRNA that just gets degraded by nonsense-mediated decay (or, theoretically, even a gene with a deleterious gain-of-function mutation, though I've never heard this raised as a serious prospect).

HDR is very precise, but it has a very low (<10% of modified alleles) efficiency, and it is only active in cells that are actively in the process of dividing, making it useless for introducing therapeutic genes into mature neurons or heart and skeletal muscle cells, and for substantial numbers of cells in organs like the liver and the skin at any given time.

Robert Church at April 11th, 2016 2:42 PM: [Pessimists] totally ignore what is happening NOW in the field. This, IMO, includes AI (albeit soon), biotech, nanotech (also, soon), making better sense of the genome with big data (happening now), the immunology tech to fight cancer(I really thought Carter would be gone by now, especially at his age), and CRISPER gene therapy.

It's quite a differnt thing, you must agree, to revise forecasts based on actual technological developments that occur in the period between when a projection is made and a future interim period, and to do so based on developments that are still undeveloped but anticipated. And for what it's worth, I don't think any of these cases should be used for either purpose. While "big data" on genomics is useful for optimizing the use of conventional medical therapy, it really doesn't do anything for either development or application of rejuvenation biotechnology — except inasmuch as it might help to optimize individual patients' regimen of rejuvenation treatments after we have enough patients already being treated for enough years to get longer-term health trajectories out of them, and also have the diagnostic assays needed to acquire such data in the first place and then use them to guide the treatment of patients. Nanotechnology of the sort that would be useful to develop rejuvenation biotechnology (ie, Drexlerian atomically precise manufacturing devices that can be used in situ in the aging body) is still many decades off into the future, with a few exceptions involving much simpler tech like advanced nanomaterials for use in scaffolding for tissue engineering: it is likely to play a key role in future iterations of the "damage-repair" heristic, but is unlikely to play any role in the first. And see above on the CRISPR/Cas9 system.

Posted by: Barbara T. at April 9th, 2016 6:02 PM: I wish that de Grey revised his forecast downwards since he has been saying that aging will be under decisive control in 25 years for at least a decade.

Dr. de Grey has on occasion in general terms speculated on how much recent advances have shortened the remaining timeline, granted that we remain far short of the needed targeted funding of rejuvenation biotechnology: Antonio has already linked two examples, and I will add a third, more detailed one:

Are Aubrey de Grey's estimations for the advent of SENS changing over the time he makes them?[Q:] In 5 years since creation of the SENS Foundation, dit the years-from- now estimation for "longevity escape velocity" shorten?[A] Aubrey de Grey, Biomedical gerontologist, Chief Science Officer of SENS Foundation:… It's been about ten years since I started making predictions … I think we're about three years closer to [robust mouse rejuvenation]; in other words we've slipped by about seven years. We've probably slipped by only a couple of years for RHR … [T]he only area in which I was overoptimistic was with regard to the ease of securing adequate funding: I think I would indeed have predicted that the funding we've actually had would have resulted in progress being about three times slower, which indeed it has been. The conclusion is clear: the world is spurning the opportunity to make a really big difference to how soon we bring aging under medical control and to save a vast number of lives, probably in the hundreds of millions.

Indeed. The vast majority of biomedical research dollars — from venture capital, Big Pharma, charitable, and the all-important public health research institutes of the advanced economies (NIH, CIHR, MRC, Australia's NHMRC, etc) — are still being ploughed overwhelmingly into the old metabolic "risk-factor" model. The advent of RHR will remain a moving target until that fundamentally changes."

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Personal fund raising lessons from the trenches

So I recently launched a 200 pound fundraiser for SENS.

Some lessons:

1. People don’t have time or energy to read walls of text about a subject (anti aging medicine) like supporters of the cause who are interested in it do.

2. If people haven’t heard of the cause they won’t donate.

3. People will generally donate because they want to stay in contact with a person, not because they care about the cause. If you are a sick and broken down person like me, there is not any goodwill to call on.

Some possible solutions:

1. Having the ability to sponsor specific projects rather than a nebulous organization like SENS.

2. Pretty infographics might make a difference (or might not). All kick-starter campaigns must have a video for a reason. Maybe some videos like those of Youreka science on youtube would help?

Fightaging.org’s blog post: Investigating the Role of Monocytes in Inflammaging

Today I’ll point you to an interesting paper in which the authors investigate one of the contributing factors that cause ever greater levels of chronic inflammation to accompany aging, in this case the factor being detrimental changes in the behavior of monocyte immune cells in older individuals. The researchers demonstrate that monocytes are influenced by rising levels of the inflammatory cytokine TNF, and either removing TNF or the problem monocytes improves the impaired immune response in aged mice.

The immune system is enormously complex, one of the many aspects of our biology in which the high level sketch – information that fills books in and of itself – must still be painted in at the detail level. One measure of what is left to learn is the present lack of understand of the root causes of many autoimmune diseases, intricate failure modes in the regulation of immune activities in which immune cells attack healthy tissue. If the research community understood the immune system completely, the scientists involved would also understand autoimmunity well enough to prevent it and reverse it. As it is, there are only hints and connections made in a growing, ceaseless river of data, while cures are yet to be found. The situation for the aging of the immune system is much the same. A variety of theories backed by differing levels of evidence explain why the immune system in aged individuals becomes both progressively less effective and progressively more active at the same time, chasing its tail to no good end. Greater activity means more inflammation, and sustained inflammation is a source of tissue damage and cellular dysfunction, a potent contribution to the pathology of many age-related diseases. Researchers have given the name “inflammaging” to this unfortunate end stage of the immune system and its detrimental effects on health.

Today we are at a point at which the research community can present a convincing story of immune aging based upon processes such as the atrophied thymus reducing the supply of new immune cells, the limited number of immune cells increasingly consisting of those uselessly devoted to a few persistent pathogens rather than capable of dealing with new threats, and so on and so forth. The relevance and importance of these processes can still be argued, however. Given the pace of progress in biotechnology, I believe that the proof of theories on immune aging will be provided by therapies capable of addressing the causes of immune decline, and this will happen long before proof can be provided via a full mapping of the biochemistry and processes of the immune system. Therapies that work will point the way, and the cost of testing any given hypothesis in mice continues to fall year after year.

Studying ‘inflammaging’: Monocytes, cytokines, and susceptibility to pneumonia

Researchers are interested in how the immune system ages. In this study, they focus on monocytes, immune cells that are central to the process of inflammation. Monocytes multiply and mature in the bone marrow and circulate in the blood stream. They are recruited to sites of injury or infection and there turn into macrophages that ingest pathogens, infected cells, or cellular debris. Monocytes are also potent producers of pro-inflammatory cytokines, small molecules that promote an inflammatory immune response.
Comparing younger and older mice, the researchers found that the latter have higher numbers of monocytes both in the bone marrow and in the blood. They also saw higher levels of TNF and IL-6, two pro-inflammatory cytokines, in blood from older mice and blood from older human donors. Studying mouse monocytes in more detail, the researchers found that the increase in TNF levels that occurs with age causes premature release of immature monocytes from the bone marrow into the blood stream. When stimulated with bacterial products, these immature monocytes themselves produce more inflammatory cytokines, thus further increasing levels in the blood.
The researchers then infected younger and older mice with the bacteria Streptococcus pneumoniae, which causes so-called pneumococcal pneumonia. They found that, although the older mice had higher numbers of monocytes in the blood and at the sites of infection, their monocytes were not able to clear the bacteria and successfully fight the infection. However, when the researchers used drugs or mouse mutations that reduced the number of monocytes or removed TNF, they were able to restore antibacterial immunity in aged mice. The researchers conclude that “monocytes both contribute to age-associated inflammation and are impaired by chronic exposure to the inflammatory cytokine TNF, which ultimately impairs their anti-pneumococcal function.” They go on to suggest that “lowering levels of TNF may be an effective strategy in improving host defense against S. pneumoniae in older adults.”

TNF Drives Monocyte Dysfunction with Age and Results in Impaired Anti-pneumococcal Immunity

As we age, levels of inflammatory cytokines in the blood and tissues increase. Although this appears to be an inevitable part of aging, it ultimately contributes to declining health. Epidemiological studies indicate that older adults with higher than age-average levels of inflammatory cytokines are at increased risk of acquiring, becoming hospitalized with and dying of pneumonia but how age-associated inflammation increased susceptibility to was not entirely clear. We demonstrate that the increase in the inflammatory cytokine TNF that occurs with age cause monocytes to leave the bone marrow prematurely and these immature monocytes produce more inflammatory cytokines when stimulated with bacterial products, thus further increasing levels of inflammatory cytokines in the blood. Furthermore, although old mice have higher levels of these inflammatory monocytes arriving at the site of S. pneumoniae, they are not able to clear the bacteria. By pharmacologically or genetically removing the inflammatory cytokine TNF or reducing the number of inflammatory monocytes we were able to restore antibacterial immunity in aged mice.

My Comment:

What drives the rise in TNF Alpha as the mouse ages? Obivously the 7 categories of SENS defined damage, but has anyone mapped a molecular pathway from them to rising systemic TNF?

Michael Rae’s reply to my comment:

@Jim: most directly, we know that TNF-α is released by senescent cells as part of the SASP.

Oxidative stress also activates nuclear factor κB, which in turn promotes the expression of TNF-α and several other inflammatory cytokines, so age-related mitochondrial mutations are an obvious candidate.

Immune cells in the atherosclerotic artery may also produce TNF-α, which may even be locally beneficial in the earliest stages of lesion development even if it has deleterious long-term local and systemic effects. As usual, looking for a solution involving up- or downregulating such production is therefore fraught.

And the rising inflammatory tone with aging can certainly be linked with lifelong dysregulation of the immune system, although probably not directlty linked with the accumulation of anergic T-cells (which, indeed, fail to produce TNF-α upon antigenic stimulus, which is part of why they’re called “anergic” in the first place).

These are all really secondary questions, however. The best way to identify the sources and relative priority of cellular and molecular damage of aging driving increased levels of TNF-α or any other alteration in the systemic environment is to clear them out and see what happens.

Fightaging.org’s blog post: Scores of Labs Should be Gearing Up to Work on Glucosepane Cross-Link Breakers, But Are They? – Michael Rae’s reply to my comment

The original post on fightaging.org

As we age skin and blood vessels lose their elasticity. People care too much about the skin and too little about the blood vessels, but that is always the way of it. Appearance first and substance later, if at all. Yet you can live inside an aged skin; beyond the raised risk of skin cancer its damaged state arguably only makes life less pleasant, and the present state of medical science can ensure that the numerous age-related dermatological dysfunctions can be kept to a state of minor inconvenience. Loss of blood vessel elasticity, on the other hand, will steadily destroy your health and then kill you. Arterial stiffening causes remodeling of the cardiovascular system and hypertension. The biological systems that regulate blood pressure become dysfunctional as blood vessels depart from ideal youthful behavior, creating a downward spiral of increasing blood pressure and reactions to that increase. Small blood vessels fail under the strain in ever larger numbers, damaging surrounding tissue. In the brain this damage contributes to age-related cognitive decline by creating countless tiny, unnoticed strokes. Ultimately this process leads to dementia. More important parts of the cardiovascular system are likely to fail first, however, perhaps causing a stroke, or a heart attack, or the slower decline of congestive heart failure.

From what is known today, it is reasonable to propose that the two main culprits driving loss of tissue elasticity are sugary cross-links generated as a byproduct of the normal operation of cellular metabolism and growing numbers of senescent cells. Elasticity is a property of the extracellular matrix, an intricate structure of collagens and other proteins created by cells. Different arrangements of these molecules produce very different structures, ranging from load-bearing tissues such as bone and cartilage to elastic tissues such as skin and blood vessel walls. Disrupting the arrangement and interaction of molecules in the extracellular matrix also disrupts its properties. Persistent cross-links achieve this by linking proteins together and restricting their normal range of motion. Senescent cells, on the other hand, secrete a range of proteins capable of breaking down or remodeling portions of the surrounding extracellular matrix, and altering the behavior of nearby cells for the worse.

The most important cross-linking compound in humans is glucosepane. Our biochemistry cannot break down glucosepane cross-links, and as a result it accounts for more than 99% of cross-links in our tissues. This isn’t a big secret. Given this you might expect to find researchers working flat out in scores of laboratories to find a viable way to break it down. After all here we have one single target molecule, and any drug candidate capable of clearing even half of existing cross-links would provide a treatment that can both reverse skin aging and vascular aging to a much greater degree than any presently available therapy. The size of the resulting market is every human being, the potential for profit staggering. Yet search on PubMed, and this is all of relevance that you will see published on the topic in the past few years:

– Preferential sites for intramolecular glucosepane cross-link formation in type I collagen: A thermodynamic study.
– Glucosepane and oxidative markers in skin collagen correlate with intima media thickness and arterial stiffness in long-term type 1 diabetes.
– Skin advanced glycation end products glucosepane and methylglyoxal hydroimidazolone are independently associated with long-term microvascular complication progression of type 1 diabetes.
– Glucosepane: a poorly understood advanced glycation end product of growing importance for diabetes and its complications.
– The association between skin collagen glucosepane and past progression of microvascular and neuropathic complications in type 1 diabetes.

This is a tiny output of work. The research and development world is not beating a path here as it should. The thesis is that this lack of enthusiasm exists because the state of tools and processes needed to work with glucosepane has long been somewhere between underdeveloped and nonexistent. No group will choose to work in an area in which they have to build the tools first when there are so many other choices available. This sort of chicken and egg situation exists in numerous places in every field of science and technology, small fields where a great deal might be achieved, but no-one does anything because the short-term rational choice is to do something else in an area where the tooling already exists. This is why we need advocacy and philanthropy, to fix problems of this nature. In recent years the SENS Research Foundation has been funding development of the tools needed for research groups to work with glucosepane in living tissues, and just this year we have seen the first published results: a simple, cheap, efficient method of creating as much glucosepane as needed for ongoing cell and tissue studies. There is now no roadblock standing in the way of any researcher wanting to run up batches of glucosepane, create small sections of engineered skin and blood vessel tissue, generate cross-links in that tissue, and then carry out assessments of drug candidates for clearing those cross-links.

The tools are a big deal, I think. Glucosepane clearance is a very narrow, very small pharmacological problem with a huge pot of gold on the other side. Pharmaceutical companies and established laboratories should be packed with staff running, not walking, to work on this. It is crazy that anyone has to be out there banging the drum to draw attention.

My Comment:

Um, there still aren’t any validated antibodies to act as markers of glucosepane removal in vivo. Surely that is a missing significant tool?

Jason Hope donated half a million dollars a few years ago to set up a SENS lab at Cambridge headed by Dr William Bains. I don’t know if this money has now run out, or if they are still getting some research done out of it (probably by working largely for free).

I saw mention somewhere (that I can’t remember) that they tested a series of supposed marker antibodies against glucosepane and found that none of them were very specific and useful.

Could the SENS Foundation run another kickstarter campaign on Lifepan.io in the new year to raise money for the Cambridge Lab to generate antibodies to glucosepane and then validate them? How much would this cost? Would $45k be enough, or is this a more expensive undertaking?

Michael Rae’s reply to my comment:

@Jim: you’re quite correct that anti-glucosepane antibodies are a critical tool, and you’re remembering rightly that the Cambridge SENS Lab found that none of the commercially- or academically-available putative anti-AGE antibodies were any good at this job. In fact, it’s worse than that: none of the putative anti-AGE antibodies are really any good for detecting any AGE at all, and barely serviceable against related adducts like CML! We shut down the Cambridge lab (indeed, Dr. Bains fell on his sword) largely because without this basic tool, they had no way to carry their research forward.

Happily, the successful and convenient synthesis of glucosepane by our Yale researchers has enabled them to start working on developing this tool. They are now working to incorporate glucosepane into synthetic, chemically-uniform crosslinked peptides, which can then be used as antigens with which to vaccinate rabbits. Once vaccinated with these antigens, the animals are expected to generate antibodies targeting glucosepane-containing peptides. The B-cells that generate the antibodies in such animals can then be isolated and immortalized, generating monoclonal glucosepane-targeting antibodies on an industrial scale.

@Steve H: I appreciate your reasons for wanting to go with IGG. Consider, however, the longer-term advantages to the entire biomedical gerontology community of using the Lifespan.io platform: by helping to build up a roster of projects and a track record of success, they can attract more projects and eventually begin digging more aging research studies out of the woodwork, raising the profile of the entire sector and eventually allowing Lifespan.io to use their royalties for their advocacy activities for the advancement of anti-aging research.

Fightaging’s post: The Long Road Ahead to Exercise Mimetics – Michael Rae’s Reply to my comment

The original post on fightaging.org

Today I’ll point out a couple of recent research publications on the topic of the molecular mechanisms of exercise: how it might work to improve health, how it extends healthy life span but not maximum life span in animal studies, and how the response to exercise might be safely improved or otherwise manipulated. Researchers nowadays tend to comment on future directions for drug discovery based on their investigations of exercise, and in that this slice of the field is becoming much like calorie restriction research ten to fifteen years ago.

Take a moment to think about how much work and funding has gone into investigations of calorie restriction and the search for drug candidates that can mimic even just a fraction of the beneficial metabolic alterations and extension of healthy life spans that occur in response to calorie restriction: probably a few billion dollars and year after year of dedicated investigations by hundreds of scientists in the past decade alone. Yet at the end of all that, and after the collection of enormous amounts of data, there is only a small number of drug candidates, few of which are anything other than marginal in animal studies, none of which can reproduce all of the beneficial changes observed in calorie restriction, and there is still no comprehensive accounting of how calorie restriction works under the hood, just an outline of ever-growing complexity.

It has taken fifteen years to get that far. Processes like the reaction to restricted calorie intake and exercise are enormously complex. They impact near every aspect of metabolism and cellular biology, and the quest to understand them well enough to manipulate them is more or less the same thing as the quest to understand cellular biology completely. This and the past history of calorie restriction mimetic drug research is why I’m not holding my breath waiting on exercise mimetic drugs. Researchers will talk about this as a goal, just as they have talked about calorie restriction mimetic drugs, but the reality is that the inherent complexity involved makes this is a very long-term project, one that tends to produce marginal outcomes at great expense. Exercise mimetics and calorie restriction mimetics that are safe and reliable would be a pleasant thing to have around, to be sure, but it seems to me that at the present time there are better and more cost-effective approaches to the treatment of aging as a medical condition.

Exercise Pills: At the Starting Line

“Excessive caloric intake and limited physical activity contribute to the current explosion of ‘modern’ chronic diseases such as obesity, type 2 diabetes, muscle atrophy, and cardiovascular diseases. By contrast, regular physical exercise maintains glucose homeostasis and induces physiological adaptations that effectively prevent, and often reverse, these diseases. Recognizing the human and economic burdens these diseases cause, and taking into account the health benefits of exercise, have led many exercise scientists to suggest that physical exercise may be the preferred method in the treatment and prevention of these ‘modern’ chronic diseases.
Unfortunately, exercise compliance levels are almost universally low, especially for people using home-based exercise programs. A variety of factors including poor physical condition, weakness, sickness, lack of time, and poor motivation contribute to low exercise compliance. The much publicized poor compliance begs the question: is there an alternative approach that both induces the health benefits of physical exercise and overcomes the problem of low compliance rate? Regular physical exercise activates a number of molecular pathways in whole organ systems and reduces the risk of developing numerous chronic diseases. Although nothing can fully substitute for physical exercise, candidate exercise pills that have emerged in recent years may be an attractive alternative.”

Exercise in a bottle could become a reality

“Researchers exposed a thousand molecular changes that occur in our muscles when we exercise, providing the world’s first comprehensive exercise blueprint. “Exercise is the most powerful therapy for many human diseases, including type 2 diabetes, cardiovascular disease and neurological disorders. However, for many people, exercise isn’t a viable treatment option. This means it is essential we find ways of developing drugs that mimic the benefits of exercise.” The researchers analysed human skeletal muscle biopsies from four untrained, healthy males following 10 minutes of high intensity exercise. Using a technique known as mass spectrometry to study a process called protein phosphorylation, they discovered that short, intensive exercise triggers more than 1000 changes.
“Exercise produces an extremely complex, cascading set of responses within human muscle. It plays an essential role in controlling energy metabolism and insulin sensitivity. While scientists have long suspected that exercise causes a complicated series of changes to human muscle, this is the first time we have been able to map exactly what happens. This is a major breakthrough, as it allows scientists to use this information to design a drug that mimics the true beneficial changes caused by exercise. Most traditional drugs target individual molecules. With this exercise blueprint we have proven that any drug that mimics exercise will need to target multiple molecules and possibly even pathways, which are a combination of molecules working together. We believe this is the key to unlocking the riddle of drug treatments to mimic exercise.”

My Comment

@Ham – Dave Sinclair’s sirtins do have repeatable results in mice. None of the 5 underfunded SENS technologies have this. Senescent cells only have one published paper in a mouse model with a shortened lifespan. The use of enzymes to clear oxidised LDL from the lysozome has only been demonstrated in a dish (although Jason hope has put his money behind this to some unknown degree). Mitosens has only been demonstrated in the dish except for one gene (ND4). And breaking gluscospane has not even been demonstrated yet.

Demonstrate one of the above in a decent mouse model a few times with a health o lifespan benefit and a biotech or pharma may come knocking.

Michael Rae’s reply to my comment:

All:

Posted by: Jim at October 6, 2015 6:56 PM: None of the 5 underfunded SENS technologies have [repeatable results in mice]. Senescent cells only have one published paper in a mouse model with a shortened lifespan. The use of enzymes to clear oxidised LDL from the lysozome has only been demonstrated in a dish (although Jason hope has put his money behind this to some unknown degree). Mitosens has only been demonstrated in the dish except for one gene (ND4). And breaking gluscospane has not even been demonstrated yet.

Hang on … By “None of the 5 underfunded SENS technologies,” I take it that you mean everything except cell therapy and extracellular aggregate clearance. Those two are indeed substantially better-funded than the other planks in the platform, but there is relevant work in rodents for several of the other planks.

First, you’ve yourself already mentioned the proof-of-concept clearance of senescent cells from aging tissues of hypomorphic BubR1 mice. You’re right to call out that this is a quite artificial model, but I’ll remind you that the field was jumpstarted by that work rather than ending with it. I’ll remind you that Julie Anderson from the Buck Institute presented thrilling results using Judith Campisi’s unpublished system (nothing of direct human translatability) in a Parkinson’s disease mouse model at SENS Research Foundation’s Rejuvenation Biotechnology 2014 conference, and the system has now been shown to prevent or reverse a range of diseases of aging modeled in transgenic mice. Additionally, as you know, Kirkland and van Deursen have demonstrated that ablation of senescent cells improves aging phenotypes in wild-type mice.

Alagebrium appears to provide quite multiple in vivo proofs-of-concept for rejuvenating tissues by breaking AGE crosslinks: it reversed age-related increases in myocardial stiffness in dogs, and reduced vascular stiffness and improved left ventricle function in nonhuman primates, and did multiple wonders in diabetic rodents. Its mechanism of action remains unclear, and its direct human translatability is demonstrably zero, but it almost certainly involves glycation and is widely thought to break medically-important crosslinks in these species.

As you parenthetically allude, bit more than 5 years ago, just prior to the formal institutional division, SRF/MF funded research on rendering mitochondrial mutations harmless reversed blindness induced by allotopic mitochondrial DNA mutations in rats. Yes, it’s only one gene, and yes, it’s rescue of an AE-induced harm, so it’s neither as exciting nor as conclusive a demonstration of as we would all like. But it ain’t mechanically-disaggregated hepatocytes 😉 .

As regards intracellular aggregates: while we usually associate vaccine-based therapies with clearance of extracellular aggregates, several vaccines targeting beta-amyloid protein clearly clear intracellular aggregate in the process, and some of the tau-targeting vaccines do the same.

And while it’s a highly incomplete solution (because it’s limited to delivering more of an existing lysosomal enzyme rather than giving the lysosome a novel, engineered enzymatic capacity to degrade intracellular aggregates, we discuss an early proof-of-concept for prevention of atherosclerosis through enhancing the macrophage lysosome in mice in Ending Aging.

The examples start to get less impressive or direct after that, but let’s remember that the “damage-repair” heuristic of SENS has undergone substantial in vivo validation already.

Posted by: Slicer at October 7, 2015 1:35 PMJim – “It works in mice” isn’t a very good qualifier here, because the metabolic differences between humans and mice often involve longevity. Sirtuins could simply be getting these mice up to the human level. Turns out that it’s really, really easy to increase the lifespan of a fruit fly and somewhat easy to increase the lifespan of a mouse.

I don’t think the analogy holds: the main issue with invertebrate models is that they have entirely different body plans and life histories than mammals, and age in a dramatically different way. C. elegans’ mature bodies are composed entirely of cells that don’t divide, so they don’t develop cancer; they don’t have hearts or circulatory systems as they occur in mammals and thus don’t suffer heart disease or atherosclerosis; they don’t live long enough to accumulate mitochondrial DNA deletions, or several other key forms of molecular and cellular damage that contribute to aging in mammals; some investigators believe that they almost always die of starvation due to failure of the muscles in their pharynx; and they have the capacity to enter into the “dauer state,” a kind of deep suspended animation, when challenged with food withdrawal or a range of other stressors, which likely confounds any data on extended fasting periods that (my esteemed mentor to the contrary) is really not analogous to rodent or human CR.

Many, many small molecules extend lifespan in these organisms by activating stress pathways or quenching free radicals; so far, none of these chemicals do so in normal, healthy mice. True, even studies in mice don’t always translate directly to humans — look at all the failed cancer drugs that cure the disease in mice — but they’re a much better start!

Posted by: Slicer at October 7, 2015 1:35 PMThe SENS approach to the obesity epidemic is the same as its approach to the heroin epidemic and the [faster methods of] suicide epidemic. It’s not in the business of stopping people from ending themselves, whether they use guns, needles, or cheeseburgers.

Of course, we aren’t and won’t be in the literal suicide prevention business (and have no intention of attempting to mandate that people accept rejuvenation therapies, but people who become obese aren’t suicidal, nor resigned to premature sickness and death. And while it won’t address the purely aesthetic aspects of obesity, rejuvenation biotechnologies will certainly prevent, arrest, and reverse the diseases and debilities that are caused by the metabolic derangements of obesity, which are (after all) only earlier-onset, selective exacerbation of the byproducts of normal metabolism that drive the disease and debility of aging.

In addition to repair, removal, replacement, and rendering harmless aging damage directly induced or accelerated most directly by excessive visceral adiposity (notably, ablation of excessive visceral adipose tissue macrophages and senescent preadipocytes, to reduce systemic inflammation and restore hepatic insulin sensitivity), the same rejuvenation biotechnologies that will eradicate atherosclerosis, clear out senescent cells, repair damaged joints, replace pancreatic beta-cells destroyed or rendered dysfunctional late in type II diabetes, replace or patch failing kidneys, repopulate failing hearts with functional cardiomyocytes while eliminating the hypertension that drives hypertrophy and dysfunction, etc.

And yes: we’re going to make people’s bodies invulnerable to cancer, whether a woman is on a course toward breast cancer comes from being made obese during her developmental window, or because she worked shift work as a nurse caring for people when aging ravages their bodies, or because she struggled with alcohol, or for no discernible reason other than the normal operation of the machinery of a body always balancing development and tissue renewal against the risk of out-of-control cell growth.

The SENS approach to the obesity epidemic is thus the same as it is for the aging epidemic. The metabolic drivers don’t matter: repairing the damage matters.

Fightaging’s post: A Review of Work on Targeting α-synuclein Aggregates

The original post on Fightaging.org

Here I’ll point out a recent review of approaches to treat one of the more common synucleinopathies, conditions related to – and thought to be caused by – the abnormal accumulation of α-synuclein in tissues. The pathologies of numerous age-related diseases are linked to various different types of protein aggregate that are observed to build up with age: misfolded or simply overabundant proteins that precipitate to form solid clumps and fibrils. Amyloids are well known for their association with Alzheimer’s disease, but there are many types of amyloid and many corresponding amyloidosis conditions. Similarly tau aggregates are linked to the tauopathies. The list goes on, and of course includes α-synuclein.

Why do these various different aggregates appear in old individuals but not young ones? Most of the evidence to support various theories comes out of Alzheimer’s research, as that field has far more funding and far more scientists working on the problem. Amyloid levels in the brain are dynamic on a fairly short timescale, and the buildup of amyloid has the look of slowly failing clearance mechanisms. These might include general dysfunction in the choroid plexus filtration of cerebrospinal fluid, or in the drainage channels that carry away metabolic waste from the brain, or the mechanisms of the blood-brain barrier intended to shunt unwanted waste out of the brain and into the blood system. These and related forms of dysfunctions could plausibly arise from many of the forms of cell and tissue damage thought to cause aging, or from their consequences such as inflammation, loss of muscle strength, loss of tissue flexibility, and so forth.

The most promising near term approach to protein aggregates is to build treatments than can be periodically applied to clear out the buildup. Immunotherapies are so far the best of ongoing efforts, enlisting the immune system to attack and break down the aggregates, but there is still a way to go towards robust and reliably outcomes. Clinical trials have so far been disappointing, as is often the case in the first round of attempts in any area of medicine. Equally, other classes of rejuvenation therapy will be needed to repair the problems in clearance of aggregates that cause the buildup in the first place: just getting rid of the aggregrates themselves isn’t a full solution, just a much better class of sustaining treatment than is presently available.

Work on clearing α-synuclein runs in parallel to work on amyloid-β, and with the same general pattern of progress, in that immunotherapies look to be the best path forward for now, yet only incremental benefits have been shown to date via this appreach. This review is focused on Lewy body dementia, but the approaches to clearing α-synuclein might be applied to any of the other synucleinopathies, such as Parkinson’s disease.

Disease-modifying therapeutic directions for Lewy-Body dementias

On Persuasion as Activism for Rejuvenation Research

https://www.fightaging.org/archives/2015/07/persuasion-as-activism-for-rejuvenation-research.php

My comment:

To be honest I don’t talk to my friends and family about the prospects for life extension technologies, I don’t know how to do it without sounding like a maniac.

I’m thinking the best way I could get them to contribute would be to try and do a fundraising activity (run a marathon or something) to try and raise $2,000, and tell them the money will go towards mitochondrial research, which may then lead to much longer and healthier human lifespans. Or glucospane cross link breaking enzymes, which may lead to more youthful looking skin “Would you like to have younger looking skin? Then sponsor my attempt to raise $1,000 for research into enzymes that break collagen sugar cross links”.

I can’t afford to join the 300 and pay $1,000 per year for the next 25 years (I am sick and working a simple minimum wage job at the moment). But perhaps I could raise some money from my social network, and at the same time get some of my friends actually thinking about the prospects of aging research.

Only problem with that is that I currently have severe allergic respiratory problems that make me bedridden. So the above plan (if it actually makes sense in real life) is on hold.

First things first 1: I need to get my health back, which hopefully going on a clinical trail by the end of the year (and not winding up in the placebo group) will do.

2: Do a rock climb/run time/weight life goal/visit X towns on the coast goal. Try to raise $1,000.

3: If that works try to do it every year.

4: Set up a site or some tools to enable others to do the same thing (answers to FAQs like “why sponsor this research? Isn’t the government paying for it?”).