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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