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Life Extension Magazine

Human Age Reversal at Harvard University

William Faloon
William Faloon

When I incorporated the Life Extension Foundation, I envisioned a time when human longevity would not be constrained to a finite number of years.

I was confident technology would emerge to enable science to gain control over pathological aging. When this biomedical turning point occurs, healthy life spans will extend beyond what anyone imagines today.

Over the past two years, our hypotheses in the 1970s have emerged into scientific probability. I am pleased that Life Extension® was able to contribute in a small way to an emerging gene editing technique that may enable age reversal to transform soon into clinical reality.

"Editing" Our Human Genome In Vivo

As we age, genes that maintain cellular health and vitality are down regulated, while genes that promote disease and senescence become overexpressed.

Once physicians are able to regulate or "edit" cellular genes, then youthful health may be restored to the entire individual.

Articles in this month's issue of Life Extension® magazine describe a technology called CRISPR that has been developed and is being improved and extensively used at Harvard University and other institutions.

Although most readers will find it difficult to comprehend, what's important to know is that CRISPR (clustered regularly interspaced short palindromic repeats) also offers a new way to rapidly transform senescent cells to regain youthful function and structure.

CRISPR/Cas is a DNA cutting system originally developed in nature by bacteria as a way to destroy the DNA of viruses that frequently attack them. A natural version of CRISPR has been adapted by scientists to enable the reprogramming of cellular DNA to rid cells of unfavorable genetic changes. Once perfected, old cells may be rejuvenated and never age again.

Programming Our Genes Like Computers

Programming Our Cellular Genes like Computers  

The CRISPR/Cas system is empowering scientists to do very controlled gene editing, which means adding, disrupting or changing the sequence of specific genes. This has led to exciting new methods of transiently or permanently modifying gene action, either to increase or decrease the activities of targeted genes in a controllable way, potentially anywhere in the body and anywhere in one's complete set of genes and DNA (our genome).

Since key features of aging are powerfully controlled by how genes are activated or inactivated (expressed or suppressed) in the body, these are critically important developments.

Introducing the Harvard Pioneer of CRISPR

Dr. George Church is a pioneer in the area of genome engineering and the development of gene editing tools based on the CRISPR/Cas9 system (referred to as CRISPR here).
Dr. Church has already been able to reverse aging in human cells using CRISPR technology, and expects the first clinical trials of this technology to begin within as little as one year.

In response to these breakthroughs, Life Extension®magazine sent Dr. Gregory M. Fahy to Harvard University to interview Dr. Church. We needed to clarify the opportunities for reversing human aging to save the lives of most of those reading this article now.

These articles/interviews are written to enlighten our scientific supporters about this new age reversal modality. All readers should appreciate that this novel technology is being developed for the purpose of rapidly integration into the human clinical setting.

Opening Comments by Dr. Greg Fahy…


Is the End of Aging near at Hand?

Gregory M. FAHY, PhD
Gregory M. FAHY, PhD

As a student of the aging process, I have been attending scientific meetings devoted to aging since the early 1980s, and have seen and heard a lot of very exciting things. But when I attended George Church's talk at a conference sponsored by Aubrey de Grey's SENS Foundation near the end of 2014, I realized that I had just heard the most remarkable talk in my life.

Why? For three very simple reasons.

First, as Dr. Church's talk highlighted, aging seems to be controlled to a large extent by the action of a rather small subset of your genes, and especially by master genes that control large numbers of other genes. Your genes, of course, are areas of your DNA that determine your eye color, your hair color, your sex, your height, and other characteristics of your body. But what is becoming increasingly clear is that genes also determine how you age—and maybe even whether you age.

Second, Dr. Church described how technologies have advanced to the point where the activity of your genes—whether the genes are "turned on" (expressed) or "turned off" (repressed, or down regulated)—can increasingly be controlled. And this is not happening in just a test tube, but in whole bodies, and even in the brain.

Dr. Church's focus is on CRISPR (clustered regularly interspaced short palindromic repeats) technology, which is a relatively new and particularly powerful method for adjusting gene activity in many different ways.

CRISPR can "edit" or change genes for the purpose of correcting deleterious mutations, or to create deliberate mutations that can have positive effects (such as in knocking out the effects of pro-aging genes). So the implication is very clear: If aging is controlled by master genes, and if the activity of such genes can now be intentionally controlled, then we are beginning to approach the control of aging on a very fundamental level. And the same technology can be applied to the correction of many diseases as well, whether age-related or not.

Finally, it would be of no use just to have the power to control aging if there was no will to utilize that power and move aging control to the clinic. Fortunately, Dr. Church wants his achievements to be rapidly translated into the clinical arena. He wants to make the control of aging a practical reality—and soon. And Dr. Church, as a highly distinguished professor of genetics and major figure at Harvard Medical School, is in an excellent position to make his wishes come true.

In an interview with the Washington Post at the beginning of December 2015,1 Dr. Church said that his lab is already reversing aging in mice, and that human applications may only be a few years away. Dr. Church stated:

"One of our biggest economic disasters right now is our aging population."
"If all those gray hairs could go back to work and feel healthy and young, then we've averted one of the greatest economic disasters in history."1

He said he sees:

"A scenario [in which] everyone takes gene therapy, not just curing rare diseases like cystic fibrosis, but diseases that everyone has, like aging."1

Dr. Church also described his personal passion in reversing human aging when he stated:

"I'm willing to become younger. I try to reinvent myself every few years anyway."1

This new CRISPR technology may change the world, and our lives, as we know them.

CRISPR is a technology originally developed by nature to fight viruses by cutting their DNA. Fortunately, it has now been modified by scientists to enable them to make specific controlled changes in targeted places in DNA. Once physicians are able to regulate or "edit" the DNA medically, then they can begin to work on restoring a state of youthful health in aging individuals.

How serious is the promise of CRISPR? Consider the following:

  • A newer version of CRISPR was recently inserted into a re-engineered virus delivery system and successfully used to correct the gene defect that causes Duchenne muscular dystrophy in a mouse model by either direct injection into a leg muscle or by infusion into the bloodstream, resulting in improvements in the muscles throughout the body and even in the heart.2
  • A leading scientific journal, Science, at the end of 2015, declared CRISPR to be the "breakthrough of the year," standing above all other scientific discoveries for 2015.3
  • On January 7, 2016, Dr. Church's company, Editas Medicine, filed papers to launch a $100 million IPO, and the company is already being backed by Google Ventures and the Bill and Melinda Gates Foundation.4

In short, in my estimation, the CRISPR revolution is a game changer, with staggering implications. If it all works out, nothing is going to remain the same. The prospects are as transformative as—if not more transformative than—such revolutions as the advent of the electric light, telephones, personal automobiles, airplanes, personal computers, the internet, and cell phones. Only this time, it's not just about how you live, but whether you live, and how long you will live: your health, your longevity, and the effect that health and longevity will have on your enjoyment of life.

Will it really work? We will see. Opinions vary. Surely, there will be many tricks to learn and many twists and turns along the road ahead. And heavyweight scientist Craig Venter even says it will take 100 years to get it right. But George Church's lab is reversing aging in the laboratory today. So far, it's looking very promising, moving with incredible speed, and based on a very solid foundation of scientific observations about aging. My money is squarely on Church and others pursuing similar paths. The end of at least some critical aspects of aging may very well be near at hand.

And the Life Extension Foundation is participating in this innovative and visionary project. The Life Extension Foundation has assisted Dr. Church by providing him data from a human super-centenarian research project that it funded. As Dr. Church mentions in his interview, studying super-centenarians may offer new insights into how human aging can be scaled back, once we have the right genetic tools to take advantage of those insights.

Since the Life Extension Foundation is dedicated to improving healthy longevity, and since Dr. Church is working on pushing the ultimate limits of improving healthy longevity, with potentially open-ended possibilities ahead, this issue of Life Extension magazine features an extensive interview with Dr. Church that was conducted in his office at Harvard Medical School to enable us to present Dr. Church's work and thoughts to you.

This interview is much more technical than many readers will be used to, and some may not be able to understand all of it, but we felt it was important to bring this important research breakthrough for the benefit of Life Extension® readers in the pursuit of healthy longevity.

We hope you will be able to appreciate the substantive nature of what we think is likely to be a coming revolution that may touch your life in important ways.


Controlling Human Aging by Genome Editing

DR. George Church
DR. George Church

An Interview with George Church, PhD

By Gregory M. Fahy, PhD

Attempting to delay aging is now old hat. The new goal is to reverse it, not only in animals, but in humans. And age reversal is essential, as significant age-related disruption has already occurred in most people due to changes in our gene expression profiles.

Gene expression patterns change with age. This influences the rate at which an individual ages, and also determines what senile disorders they are likely to contract. But innovative gene-editing methods based on a unique technology called CRISPR (clustered regularly interspaced short palindromic repeats) are now being successfully harnessed for use as an age-reversal therapy for humans.

In response to these breakthroughs, Life Extension® magazine sent biogerontologist Dr. Gregory M. Fahy to Harvard University to interview Dr. George Church, who is a leading developer of cutting-edge CRISPR techniques. Here, Dr. Church explains remarkable opportunities for transforming human aging that may begin to unfold sooner than most have imagined.

This interview with Dr. Church begins with a discussion on reversing cell aging by restoring youthful gene expression.

Fahy: If aging is driven by changes in gene expression, then the ability to control gene expression using CRISPR technology could have profound implications for the future of human aging. Why do you think aging may be at least partly driven by changes in gene expression?

Church: We know that there are cells that deteriorate with age in the human body and that we have the ability to turn those back into young cells again. This means we can effectively reset the clock to zero and keep those cells proliferating as long as we want. For example, we can take old skin cells, which have a limited lifetime, and turn them into stem cells (stem cells are cells that can turn into other kinds of cells) and then back into skin cells. This roundtrip results in the skin cells being like baby skin cells.5 It's as if my 60-year-old cells become 1-year-old cells. There are a variety of markers that are associated with aging, and those all get reset to the younger age.

Fahy: That's fantastic. Does this mean that reversing skin cell aging in your face would allow you to rejuvenate your entire face?

Church: If you rejuvenate at a molecular level, it doesn't necessarily mean that everything else rejuvenates. So, for example, if my face has a scar on it, it's not going to necessarily reverse that (although theoretically it's not out of the question). But we can reverse the tendency of your cells (and therefore of your whole body) to deconstruct when you reach your life expectancy.

The Technology: How Genes and Their Expression Can Be Modified

Fahy: So CRISPR has allowed you to reverse aging in human cells. CRISPR is an exciting technology. The CRISPR molecular machine—consisting of a protein and some associated RNA—can now be made in the lab or in our own cells and can change genes and gene expression. It's extremely powerful. Please tell us more about it.

Church: CRISPR is the latest method for performing genome editing (editing of your whole set of genes). Its advantage is in part that a specific CRISPR tool can be created far more easily than other gene editing tools, and CRISPR is about 5 times more precise than other tools. The combination of the ease of construction, improved efficiency, and great flexibility makes it the most powerful gene editing tool to date. (See sidebar: Gene Editing with CRISPR)

Fahy: Right now, with CRISPR, it is possible to modify, delete, insert, activate, and tone down or completely inactivate any gene, with considerable fine-tuning, either temporarily or permanently. (See sidebar: Gene Editing with CRISPR) Now let's talk about what this fantastic new ability could be good for.

Specific Opportunities for Reversing Human Aging TFAM: Staying Energetic Indefinitely

Fahy: There are several very exciting stories in aging intervention these days. In 2013, the Sinclair lab at Harvard came out with the revelation that the aging of mitochondria (which are the producers of usable energy within cells) is driven in significant part by reduced levels of one particular molecule in the cell nucleus: oxidized NAD (NAD+).6

The team showed that they could correct mitochondrial aging just by giving old mice nicotinamide mononucleotide (NMN), which is a vitamin-like substance that can be converted into NAD+, for one week. This resulted in phenomenal overall rejuvenation, including reversal of signs of muscle atrophy, inflammation, and insulin resistance. Now your lab showed that there is a very exciting gene engineering alternative involving TFAM (Transcription Factor A, Mitochondrial). Why is TFAM important, and what have you done with it?

Church: TFAM is a key regulatory protein that is in this pathway of NMN and NAD+. It allows cells to manufacture the NMN precursor on their own, so you don't have to manufacture it outside the cell and then try to get it into the cell from outside. Ideally, you don't want to have to take NMN for the rest of your life, you want to fix the body's ability to make its own NMN and buy yourself rejuvenation for at least a few decades before you have to worry about NMN again. In order to accomplish this on a single cell level, we've used CRISPR to activate a TFAM activator, and we made it semi-permanent. (See sidebar: Gene Editing with CRISPR)

Fahy: With this technique, you were able to increase TFAM levels in the cell by 47-fold. This resulted in restored ATP levels, increased NAD+, and an increased NAD+/NADH ratio. It also increased total mitochondrial mass and reversed several other age-related changes.

Church: Yes. We have a number of ways to measure mitochondrial function and age-related losses of those functions. When we activated TFAM, these changes returned to what you would expect of a younger cell state. And we built this anti-aging ability into the cell, so it's self-renewing and eliminates the need to take pills or injections.

GDF11: Achieving Overall Rejuvenation

Fahy: Now, let's move on to GDF11 (growth differentiation factor 11), which is a protein and a type of youth factor that is present in the blood of young animals, but that declines with aging.7

Church: Yes, my lab is involved with the GDF11 story. We collaborate with Amy Wagers, a Harvard biologist famous for her work on heterochronic parabiosis, and her group, who are among the real pioneers for this.

Fahy: GDF11 has been reported to rejuvenate the heart,8 muscles,9 and brain.10 It restores strength, muscle regeneration, memory, the formation of new brain cells, blood vessel formation in the brain, the ability to smell, and mitochondrial function. All of this is done by just one molecule. Infusing young plasma, which contains GDF11, into older animals also provides benefits in other tissues, such as the liver and spinal cord, and improves the ability of old brain cells to form connections with one another.

How would you use CRISPR to make sure that GDF11 blood levels never go down?

Church: The CRISPR-regulating GDF11 could be delivered late in life, which is exactly when such an increase would be welcome. If you really wanted to stay at a certain level, you might want to put in a GDF11 sensor to provide feedback so you could automatically control GDF11 production so as to lock in a specific GDF11 level. If necessary, you could recalibrate and fine-tune this maybe once every few decades with another dose of CRISPR. But yes, it's a great molecule, and we've got a handle on it.

We are also doing a number of other projects with Amy now, dealing with a range of muscle diseases such as muscle wasting. We're working on possible treatments involving proteins such as myostatin and follistatin.

Keeping Strong Muscles and Bones

Fahy: Speaking of myostatin, the lack of which causes super-development of muscles, you mentioned in your 2014 SENS talk that you are interested in the possibility of enabling better muscle strength and less breakable bones. Is this another good treatment path for aging?

Church: Muscle wasting and osteoporosis are symptoms of aging. The key to dealing with them is to get at the core causes, even if they're complicated. There are genes known to be involved in muscle wasting and genes that can overcome that. We're interested in these very powerful things, like growth hormone, myostatin, and the target for some of the new osteoporosis drugs, RANKL (Receptor activator of nuclear factor kappa-B ligand).

Fahy: What about going beyond just correcting aging and actually super-protecting people by making them augmented with stronger bones or muscles than what they would normally have?

Church: Rather than waiting until the muscles are wasting and then trying to correct the problem, or waiting until someone breaks a bone and putting a cast on, the idea is to make the muscles and bones stronger to begin with. Think of it as preventive medicine. You have to be careful, but there are people naturally walking around with much denser bones and much stronger muscles that have no particularly bad consequences, so we know such things are possible.

Fahy: Can osteoporosis be reversed?

Church: I would say osteoporosis definitely could be reversed. The process of bone building and bone breaking down is a regulated process that responds to conditions such as the good stress of standing or running. So yes, it's an example of something that's reversible.

IKKβ: Reversing a Possible Whole-Body Aging Program

Fahy: Let's move on to another aging process of potentially tremendous significance. According to a paper published in Nature,11 body weight, bodywide aging, and longevity are all controlled to a significant extent by the overexpression of one particular protein, IKKβ, in one highly specific place, the microglial cells in the medial basal hypothalamus in the brain. When this overexpression is prevented in mice, median and maximum life spans go up by 20% and 23%, cognition improves, exercise ability improves, and skin thickness and bone density also improves. In addition, collagen cross-linking is reduced and gonadotropin output goes up. If these improvements could be combined with the improvements caused by the other interventions we have discussed, the implications could be staggering.

Church: Yes. What you're referring to is something that a certain school of thought thinks is aging programmed by the neuroendocrine system, by the brain, and the reason why mice start dying at two and a half years and bowhead whales start dying after 160 years.

Fahy: Yes. And it's a particularly interesting problem because not only is it important in its own right, but it introduces the practical issue of fixing aging changes that arise in the brain. This part of the brain is protected from most things put into the bloodstream by the blood-brain barrier. Is it possible to get CRISPR technology through the blood-brain barrier and possibly address that particular pathway or other pathways in the brain?

Church: The blood-brain barrier is greatly overstated in that there are many, many things that cross it, such as various drugs, viruses, and even whole cells. So, the answer is yes, we can deliver CRISPR across the blood-brain barrier.

Telomerase: Heading Off Brain Aging and Cancer?

Fahy: Telomerase is widely recognized as an enzyme that may prevent aging on the cellular level. But the lack of telomerase may also drive brain aging12 and cancer.13 Could CRISPR be used to replenish telomeres?

Church: Yes, that certainly is feasible.

The State of Gene Expression Is a Measure of Aging in Humans

Fahy: Would you please explain epigenetics, and comment on evidence that there is an epigenetic clock of aging?

Church: Epigenetics is essentially everything that controls gene expression. One component of epigenetics is DNA methylation, which consists of the addition of chemical entities called methyl groups to DNA at specific places. DNA methylation is important in part because it is a particularly easy component of the epigenome (the set of all epigenetic states) to measure. It turns out that DNA methylation changes with aging.14 In fact, the state of DNA methylation can predict the age of a person to within about three years.15

In principle, if you could change the biological age of a cell or of an organism to a younger state, and if those methylation sites (the sum total of which is referred to as the "methylome") are really reflective of age itself, then the methylome should change to the pattern you would expect at an earlier age. In other words, if aging itself changes, then this biomarker of aging should change in the same way. We use these methylation sites as a measure of how well we're doing in some of our studies where we're trying to get aging reversal, and it works extremely well.

DNA methylation is very good for estimating the age of a person, and it can also be changed. Even though it's always linked to chronological age in normal life, in the world of aging reversal and epigenetic tinkering, you can change it, and the change is meaningful.

Fahy: Not all 50 year olds are biologically 50. Some are biologically older and some are biologically younger. People age at different rates. Fascinatingly, these differences can be detected by the state of the methylome. If the methylome indicates a different age than your chronological age, you are really older or younger than your chronological age, and this was validated by a variety of other measures.14,16

Church: Yes, that is correct. The people who discovered the epigenetic clock of aging studied their outliers and found interesting correlations with them. There are multiple measurements for molecular level aging events, and they tend to reinforce one another. We don't know enough about connecting the dots between measures such as the methylome and aging factors such as GDF11, IKKβ, and TFAM, but if you're doing anything to reverse age, then the methylome should also reverse along with the reversal of aging.

Fahy: Apparently, the DNA methylation state gets more chaotic as we age. For example, the methylation patterns of identical twins begin to diverge over time, more aberrant patterns being associated with greater pathology. This is consistent with a recent theory that attributes the lack of aging in some species ("negligible senescence") to a relatively stable pattern of gene expression over time, and normal aging to unstable and increasingly chaotic patterns of gene expression over time.17 But if you change gene expression back to what it should be, all of that variability should be reversible, right?

Church: That's right. The variation in different parameters in any biological system increases when you get further away from the physiologically normal state. You can think of the methylation variance as another risk factor for aging and disease.

How to Quickly Discover and Begin to Correct Currently Unknown Causes of Aging on the Gene Level

Fahy: If aging is driven by changes in gene expression and those changes in gene expression can be reversed, then we need to be able to find all of the important age-related changes in gene expression as quickly as possible. How can this be done?

Church: Gene expression results in each cell having specific RNAs and proteins, and these can be surveyed. You don't necessarily have to define every single RNA in a particular cell to understand that cell, but you can, and we have in fact developed a new method to do this that allows us to see all of the tens of thousands of RNAs in a single cell at one time, and to see the RNAs in neighboring cells as well. So now we can see how different cells relate to one another in context. This new method, called fluorescent in situ sequencing, or FISSEQ,18 allows us to count all the RNAs in a cell while simultaneously counting all of the RNAs in all of the cells it touches. Plus, we get the 3D coordinates for every RNA molecule in every cell.

Fahy: That's unbelievable. How can you use this method to search for changes that are related to aging?

Church: Suppose there are two different kinds of cell, and we want to know what gene expression states make them different from one another. We can first compare the two cells using FISSEQ in order to determine the differences in gene expression between them. Next, we can pick specific differences we think cause the cells to be different cell types, and change the expression of those particular genes in either or both cells using, for example, CRISPR, and see if we can change one kind of cell into the other. Even if we don't get it right the first time, we can take many guesses as to what the important RNAs are and just how much to tweak them until we do get it right.

The same principle can be applied to any pair of cells. By comparing old cells to young cells, we can find out what makes an old cell an old cell, and how to turn an old cell into a young one.

Fahy: Fantastic.

Church: One of the problems with studying development and aging is that it takes a long time. But if we know the epigenetic state of all these different cells, no matter how many years apart they are, it only takes a few days to reprogram a cell and duplicate the effects of decades of slow change in the body, or reverse those effects. So in principle we could turn a young cell into an old one or an old cell into a young one because the only difference between them is epigenetics, or gene expression.

Fahy: What other ways are there to identify powerful gene targets for intervention into human aging?

Church: There are basically four good ways to find key gene targets.

First, we can look at genes that underlie person-to-person variability in such things as low risk for viral infections, diabetes, osteoporosis, and so forth. The most extreme example here would be to compare normal people to super-centenarians, those who live to the age of 110 or older. They might have genes that are protective enough to find even with a small number of individuals, or even with a single individual.

There are hundreds of genes that have small effects, but then way out on the end of the bell curve is something like the myostatin double null mutant or human growth hormone over/under production. Genes that have gigantic effects and completely dominate the effects of small environmental and small genetic influences are the right kind of gene to look for.

The second way to find the best gene targets is to pick from discoveries made from basic studies like the GDF11 and TFAM that we talked about earlier.

A third way is to use a specialized highly genomic strategy, such as mutating thousands of genes one by one to see if any of these mutations block aging, or using the FISSEQ method we discussed earlier.

The fourth way to identify powerful gene targets is to compare closely related animals, one of which ages much more slowly than the other (like naked mole rats vs. rats).

No matter where you get your lead, you don't have to worry about having too many hypotheses. Just use CRISPR to activate or inhibit that candidate gene and look for the biomarkers of aging reversal we discussed earlier. The idea is to see whether your change has an impact or not, and whether it acts synergistically with the other things that have been shown in the past to have an impact.

Fahy: So if we saw something unusual or provocative in super-centenarians, we could create the same change in, for example, a normal human cell line and observe whether the right longevity pattern emerged.

Church: Yes.

Fahy: I've been told by James Clement, who is being funded by the Life Extension Foundation to do collaborative work with you on the genetics of super-centenarians (See sidebar: Life Extension Foundation Funding of CRISPR Research), that you might even be able to take super-centenarian gene expression patterns and put them into mice and see if the mice age more slowly.

Church: Right. Our protocol will likely be to collect leads from the four different sources and try them out first on human cells. By going straight to human cells, we won't get into the trap of spending years working on mice, which is rather expensive, only to find out that it doesn't work in humans. We can actually do a cheaper and more relevant study in human cells, confirm them in mice, then test them in larger animals, and then in humans. I think that going from human cells to mice and back to humans is likely to save us time and money. Many human cellular testing systems are getting better and better, such as "organs on a chip" or organoids, which are getting to be more and more representative of in vivo biology.

Eliminating the Tradeoffs of Intervening in Aging

Fahy: Could the ability to target some genes and not others using CRISPR also make it possible to eliminate the side effects of some anti-aging interventions? For example, I'm working to show that it's possible to regenerate the thymus in humans and restore naïve T cell production using growth hormone. Although growth hormone does not cause cancer in adult animals or people, it slows down DNA repair in animals, which is an effect that is unrelated to the beneficial effects of growth hormone and to regenerating the thymus.

Church: So you'd like to get rid of that effect on DNA repair while keeping the good effects.

Fahy: Yes. If you can use CRISPR to go right to the genes of interest and not act through the usual pathways, which also lead to places you don't want to go, the unwanted effects should be avoidable, right?

Church: Exactly. You could make a list of all the growth hormone targets and either pick the growth hormone targets you like and activate them selectively, or pick the growth hormone targets you don't like and block them so you could use growth hormone normally without inhibiting DNA repair.

The Feasibility of Applying CRISPR Technology to the Whole Body

Fahy: To reverse human aging, CRISPR technology will ultimately have to be applied in the whole body, and not just to cells in a test tube. How feasible is it to apply CRISPR technology in the intact body?

Church: Gene therapy can be based on either ex vivo manipulations, in which cells are removed from the body, genetically modified, and then put back into the body, or on in vivo (within the body) methods, in which, for example, a modified virus might be used to carry a gene package into many different cells in the body. Each of these methods has pros and cons.

There are viral and non-viral delivery systems that could be used to deliver CRISPR constructs and that will leave the blood vessels and go into the tissues. The delivery system could contain the CRISPR plus guide RNA plus the donor DNA (See sidebar: Gene Editing with CRISPR), or it could just comprise the CRISPR, guide RNA, and protein activator, and so on. But whether it's a viral delivery or a non-viral delivery method, the total mass of gene editing devices that has to be delivered will have to be considerable. But there is no rush, you can deliver them slowly.

Fortunately, there are ways to manufacture biologicals that are dirt cheap. Things like wood and even food and fuel are all roughly in the dollar-per-kilogram range. If we could similarly make a kilogram of a viral delivery system and load it up with CRISPR, then it could become inexpensive enough to apply to the whole body.

Fahy: Yes, a kilogram would be plenty! So, the viral delivery system contains a gene for CRISPR, a separate gene for the guide RNA, etc. When it delivers these genes to the cell, the cell makes the resulting proteins and nucleic acids, and all of the components simply assemble all by themselves in the cell, is that right?

Church: Yes.

Fahy: Which is the best CRISPR delivery system?

Church: Adeno-associated viruses (AAV) are one of the favorite delivery systems right now because they can be nudged into going to tissues other than the liver (where many other delivery systems end up) more readily. This is an active field of discovery. It's moving quickly, and the CRISPR revolution just made it an even more desirable field to study.

Safety

Fahy: How selective can a virus be engineered to be for delivering CRISPR to just one cell class and not another in the body?

Church: For every thousand cells of a particular type that you target, you might deliver your payload to one other cell of a different type that was not targeted. That should be good enough. Also, if you've got something that is required for cells in general, then it should be delivered to all cells. Even if you have something that is cell specific, it doesn't necessarily matter to which cells it is delivered. But in cases where it does matter, you can get the delivery right about 999 times out of 1,000 right now.

Fahy: Could there be safety issues of having a one in 1,000 misdelivery rate? That would still come out to a lot of misdeliveries in a whole body.

Church: It helps to remember that most drugs actually go to all the cells in your body. It would be a double standard to say that CRISPR has to be more specific than any previous drug.

Safety also depends on what brand of "explosives" you're dealing with. It's like nitroglycerin versus TNT. If you make safety one of your top priorities, you're not going to be using something that can go awry, until you can make cell specificity very high.

Fahy: Another point of importance for the safety of using CRISPR in the whole body is not just which cell it goes into, but whether it edits the right gene or not. How accurately can CRISPR be targeted within the genome?

Church: In practice, when we introduced our first CRISPR in 2013,19 it was about 5% off target. In other words, CRISPR would edit five treated cells out of 100 in the wrong place in the genome. Now, we can get down to about one error per 6 trillion cells.

Fahy: This must mean that the chance of a serious error is now low enough that it is very hard to measure, and far less than the spontaneous mutation rate.

Church: Yes. And beyond this, there are ways to use small molecules as conditional activators to ensure that intended effects happen only in the correct cells. The combination of a totally safe small molecule activator and programmable targeting is unprecedented.

Other checks can be put in as well for even greater safety. For example, once a viral payload gets inside the cell, it can make further decisions. It can essentially ask, "Am I in the right place?" before it acts. There's a whole field of molecular logic circuits that could be applied in order to avoid errors.

Affordability

Fahy: Is it going to be affordable for a human to reverse his or her aging process using this kind of approach?

Church: If you look at the current price, it looks very unaffordable. There are about 2,000 gene therapies that are in clinical trials, but the only gene therapy that's approved for human use costs over $1 million per dose. You only need one dose, but at that cost it's obviously unattainable for most people. It's the most expensive drug in history, as far as I know.

Fahy: What is that drug?

Church: It's called Glybera®. It treats pancreatitis, a rare genetic disease. But sequencing the human genome used to cost $3 billion per person, and now has come down to just $1,000 per person, so I think getting from over $1 million down to the thousands shouldn't be that hard.

Fahy: Another cost saver for aging intervention would arise if we could roll back aging significantly just by modifying five to 10 genes. That might get the overall cost down to something attractive.

Church: Right. The combination of having to hit, say, a trillion cells in the whole body and 10,000 genes would be daunting. But if you can do a subset of cells and a subset of genes, then it becomes more feasible to make it affordable.

Fahy: You have said that CRISPR therapy might have the potential to replace conventional drugs. Why is that?

Church: A big advantage of CRISPR is that it offers better opportunities than conventional treatments for "putting knobs in" where there aren't any control knobs now. Right now, you have to be very lucky to have a potent drug that will do what you want it to do and nothing else. With CRISPR, we can be far more precise.

How Many Aging Corrections Can Be Made at One Time?

Fahy: If we know what to do and we can afford to do it, how quickly can we proceed to correct aging? What about simultaneous modification of, say, 10 different cell types in the body that were causing most aging changes? Could they all be modified at the same time?

Church: "All" is a big word, but I think that many could be modified at once. This could be done by what we call multiplexing, using a mixture of viruses or delivery vehicles to enable many things to be done at one time. But you can also go slowly, starting with the highest priority tissues first and then going on to lower priority areas. Determining which tissues are the highest priority could vary with the patient's heredity, which might cause a particular tissue to be at higher risk for aging faster.

Getting It to the Clinic: How Long Will It Take?

Fahy: Using your most favorable pathway for intervention, how long will it take before a human trial might be possible?

Church: I think it can happen very quickly. It may take years to get full approval, but it could take as little as a year to get approval for phase one trials. Trials of GDF11, myostatin, and others are already underway in animals, as are a large number of CRISPR trials. I think we'll be seeing the first human trials in a year or two.

Fahy: Can you say what those trials might be?

Church: I helped start a company called Editas that is aimed at CRISPR-based genome editing therapies in general.20 Some of those will be aimed at rare childhood diseases and others hopefully will be aimed at diseases of aging. We also have a company focused specifically on aging reversal that will be testing these therapies in animal and human models.

Aging Intervention, the FDA, and the Dietary Supplement Model

Fahy: Is the fact that the FDA does not recognize aging as a disease a problem?

Church: The FDA is willing to regulate many symptoms of aging, such as osteoporosis, muscle decay, heart disease, mental agility, and so forth. It tends to be harder to prove a preventative than it is to prove a drug that cures an immediately and hugely harmful disease. And actually, since the FDA doesn't want you making unjustified health claims of any kind, they would have to take responsibility for regulating any health-related condition that one might want to make claims about. It doesn't have to actually be a disease.

Fahy: It has been proposed that the FDA should just evaluate safety and not efficacy. How do you feel about that?

Church: I really like that. The Internet will probably weed out the non-efficacious. The nutritional supplement market is a perfect example of safety being all that is needed for approval. You can get a nutritional supplement on the market just based on safety, but you can't get a prescription drug on the market just based on safety. It really should be the same rule.

Fahy: The freedom to innovate and to create dietary supplements is what the Life Extension Foundation is all about. They fund all of my research in cryobiology, and they base their supplements on scientific literature. There are good effects of freedom and freedom to operate.

Church: That's true. I'm just saying that there is a double standard for the FDA. The standards for supplements are different from the standards for new prescription drugs.

Fahy: Perhaps if that were altered in favor of the standards for supplements, we'd have many more drugs and would all be a lot better off.

Church: Yes. Focusing on safety is probably the right model.

Fahy: Thank you, Dr. Church, for an amazing glimpse of the near future!

Life Extension Foundation Funding of CRISPR Research

By Ben Best

The May 2013 issue of this publication reported on how the Life Extension Foundation was funding the collection and analysis of genes of super-centenarians (people living to age 110 or older) to discover protective genes that allow them to live so long.

This funding as provided to group called Androcyte LLC's that initially consisted to CEO James Clement and his assistant, Parijata Mackey. They travelled the world to collect tissue samples from approximately 60 super-centenarians and their family members. Harvard Medical School geneticist, Dr. George Church, was collaborating with Androcyte to analyze the genes.

Since then, Dr. Church has achieved additional fame as being a co-inventor and pioneer in the new CRISPR gene-editing technology. Also since then, the Life Extension Foundation has continued to fund Androcyte to open a laboratory in California dedicated to applying CRISPR to deliver longevity genes, initially to mice. Androcyte CEO James Clement continues to work with Dr. Church in doing this research.

Androcyte currently has a colony of 300 mice, and growing. Sixty of these mice were received from the National Institutes of Aging, and are between 26 and 36 months of age—the equivalent of very old humans.

Androcyte has targeted about 25 promising longevity genes that are being tested in the mice via CRISPR/Cas9 gene therapy. Particular attention is being paid to the elderly mice to see if they can be restored to youth and good health.

To keep costs low, Androcyte purchased a one-acre property with an existing 1,500 square-foot building that is an hour's drive from costly Los Angeles. As it outgrew its initial vivarium (housing for mice), it added two office trailers to the property to provide additional vivarium and laboratory space.

In addition to Dr. Church and other expert consultants, Androcyte CEO James Clement has acquired the assistance of two new interns: Ellie Dubrovina and David Falzarao, who were referred by Aubrey de Grey's SENS Foundation. Ellie assists with the scientific work, whereas David assists with the care of the mice.

Androcyte has also received two elderly Arabian mares 28 and 30 years old (age-equivalent to 80-year-old humans) from a sanctuary. If genes delivered by CRISPR to the mice are able to restore youth and health, CRISPR delivery of those genes will be tested on the horses to show that large animals can also benefit. Success with the horses could pave the way for using CRISPR to bring better health and greater longevity to humans.


Gene Editing with CRISPR

Fahy: Just how efficient is CRISPR at editing targeted genes?

Church: Without any particular tricks, you can get anywhere up to, on the high end, into the range of 50% to 80% or more of targeted genes actually getting edited in the intended way.

Fahy: Why not 100%?

Church: We don't really know, but over time, we're getting closer and closer to 100%, and I suspect that someday we will get to 100%.

Fahy: Can you get a higher percentage of successful gene edits by dosing with CRISPR more than once?

Church: Yes, but there are limits.

Fahy: How does CRISPR edit genes?

Church: The way CRISPR works, classically, is by cutting DNA so many times in a specific place that eventually an error in DNA repair is made at the location of the cut, and you end up with a random change in the DNA—a mutation—at that location. For that kind of change, the longer CRISPR is around, the more likely you are to accomplish a random mutation, and that can be good if you want to inactivate a troublesome gene, since usually a random mutation will in fact inactivate the gene.

But newer versions of CRISPR are more interesting because they allow you to not just make cuts in DNA that get misrepaired, they allow you to splice a particular new piece of DNA, which can do new things, into your genome at a precise location. The trouble there is that it's possible the first round of treatment might have altered the site where you want to insert your new DNA in some of the cells, such that the site of interest can no longer be edited in those altered cells.

Fahy: Can you remove DNA also?

Church: Yes, you can delete a specific piece of DNA cleanly by making cuts not just in one place, but in two nearby places. The two cut ends are rejoined by normal DNA repair enzymes, and when this happens, the DNA that was previously between the cuts is lost. Now if you want to make a change in the DNA rather than a deletion, you can, instead, insert new DNA, or "donor DNA," between the two cuts. The stuff that's inserted either fixes the gene that you're trying to do therapy on or provides some new desired function.

Fahy: How do you tell CRISPR where you want it to edit the genome?

Church: Almost all the previous gene editing mechanisms require protein to find the genetic target of interest which has been the needle in the haystack. But CRISPR is a little like a programmable computer, in which you can program the genetic location of interest by making and providing what is called guide RNA. Since RNA and DNA are so similar to one another, and tend to stick to one another if their base sequences match, you can make an RNA molecule that will bind to a predetermined segment of DNA. The guide RNA binds to both the DNA target area and to CRISPR, and then CRISPR acts on the DNA it is attached to thanks to the guide RNA. The guide RNA does 95% of the genetic targeting work, and making guide RNA is really easy. Ultimately, the editing depends on four things working together: the genome, CRISPR, the guide RNA, and the donor DNA.

Fahy: Can you do more subtle things than delete or add DNA using CRISPR?

Church: Yes, a more interesting approach, in many ways, is to get rid of the DNA cutting activity of the CRISPR and attach to it an "activation domain" that activates any gene that's really close to it.

Fahy: You mean you can turn on a desired gene even without any signals from the cell that the gene should be on?

Church: Yes. The CRISPR/activation domain does all the heavy lifting, finding the needle in the haystack, the one place in 6 billion base pairs where you want it to bind, and once it binds, instead of cutting, it activates. That's it. Even if the gene is as off as can be, even if you have a cell that really has absolutely no intention of wanting that gene to be turned on (speaking anthropomorphically), you can still turn it on. In fact, the more off it is, the more impressive the induction ratio (ratio of the amount of gene activity after activation to the amount before activation) we get. We've seen induction ratios of up to 20,000 fold. This is amazing but it's fairly predictable.

Fahy: Can you use the same approach to turn a gene off or to reduce its expression?

Church: Yes. You can reduce its expression by putting in repressive domains. Of course, if you really want to reduce the expression, you can delete it. That'll get it down to zero but there's no finesse in it. There's no control. If you want to turn it up and down, if you want to be able to fully regulate it from very low to very high, then you don't want to make any irreversible changes to the gene, and so the repressor and activator domains are a good way of doing that.

Fahy: Tremendous. But how can you fine-tune the repressor or the activator to go from very low to very high gene expression?

Church: You can use activators or repressors that respond to inexpensive small molecules a bit like a rheostat, to turn gene expression up and down. There are lots of small molecules, which are totally safe and do nothing unless your target repressor or activator is around to respond to them. It doesn't even have to be a pharmaceutical, it just has to be something harmless that's not commonly part of your diet or your body, and you can make it into one of the world's most effective regulators. It can be something transient, where you eat it or inject it, it does its job, decays, and then nothing happens until the next injection. You can also regulate expression with light if you have a way of getting light into the tissue in question. Those are some of the ways that you can regulate it.


Dr. Bobby Dhadwar and Dr. Margo Monroe on Reversing Human Cellular Aging Right Now

After interviewing Dr. Church, Dr. Fahy had a chance to briefly interview two new postdoctoral fellows in the Church lab who have done the cellular aging reversal studies referred to in the Church interview. Here is the result of that interview.

Fahy: Please introduce yourselves and tell me how long you have both been in George's lab.

Dhadwar: I'm Bobby Dhadwar, and I've been here about a year and a half. I did my PhD at McMaster University, and then I came here to do my postdoc.

Monroe: I'm Margo Monroe, and I've been here a little short of a year and a half. I am from Florida, and then I went to Boston University to do my PhD in biomedical engineering, and then I came here for my postdoc, which concluded in November 2014. I am currently working in intellectual property at a law firm in Boston.

Fahy: Please tell me what you're excited about.

Monroe: What's exciting is: aging studies have been around forever, but this is different, we're figuring out how we can reverse aging.

Dhadwar: Agreed. We've shifted from trying to make a young animal live longer to rejuvenating an old animal to a younger state. This is a complete shift in perspective. When people originally thought about aging, they assumed it had a lot to do with accumulation of mutations, but that doesn't seem to be the case. Genetically, your genome is intact. There are no mutations that are causing the aging process. What's going on when you age is that you get this drift in the regulation of these gene networks so that you have a cell that's supposed to be, say, a skin cell, but then it starts expressing genes that it's not supposed to be expressing.

Fahy: So you're working on not only a different technology, but also a different concept.

Dhadwar: Right. A lot of times, when we talk to people about living a lot longer, they say, "No. I don't want to live into the hundreds. I don't want to be decrepit. I don't want to be in a state where I can't take care of myself for that many more years." But when we talk about living longer, we think of living a healthy, active lifestyle so that you're rejuvenated and you're really enjoying your life.

Fahy: You're actually biologically younger.

Monroe: Right.

Fahy: If you're biologically younger, all of these problems go away.

Monroe: It's like the slogan, "Oh, 50 is the new 30." It would just be a little bit more extreme in that regard.

Fahy: Yes. Exactly. One hundred is the new 30.

Monroe: Correct.

Dhadwar: There's been some really interesting studies just from the last year which makes it very interesting to look at epigenetics. You can actually tell the age of a person with a three- to four-year accuracy just by looking at the methylation status of a particular tissue in their body. We can actually use that as a tool now.

Monroe: To see if what we're doing works.

Fahy: So you don't have to develop a drug for human aging and then have to wait 50 years to see if it works.

Dhadwar: Exactly. We want to have a metric so we can ask, if we give someone this therapeutic, "Are they younger or older than before they took it?" We want to actually measure if we're reversing the aging process, and that's something we can actually push to the clinic.

Monroe: Right. It will be nice if, within a week, you can tell if a certain intervention is going to have success or not.

Dhadwar: Correct. We don't want to have to wait weeks, months or even years to actually see if an intervention has an effect, and then have to repeat the experiment, and then, in the end, find that it doesn't necessarily apply to humans. Our focus has been to develop a strategy that we can actually take to the clinic. Our endpoints will not include the age of death, because we're not interested in that.

Fahy: Obviously you're interested in signs of rejuvenation instead. Is it working?

Monroe: In-vitro, we're able to make a 94-year-old fibroblast look metabolically like a 22-year-old fibroblast. With just one gene. It is transcription factor A from mitochondria, a nuclear-encoded gene that transcribes the mitochondrial genome.

Fahy: TFAM?

Monroe: Yes, and it's so easy to do, too. It's amazing. We're so surprised.

Dhadwar: It's such a wonderful effect on just one simple target. Now, we're interested in combining that with other targets to actually complete the reversal process. TFAM might restore some of the age-related phenomena.

Monroe: The metabolic effects.

Dhadwar: Right. When you age, the number of mitochondria decline and the amount of energy your cells have declines. We can restore that, but that doesn't restore everything in the cell. So now we're targeting other pathways that will complement TFAM so we can have this cocktail that can actually reverse all the symptoms of aging.

Fahy: Spectacular. How many other things are you looking at for your cocktail right now?

Dhadwar: Right now, we're going after some of the major pathways known in aging. But we don't believe those might be all-encompassing, so that's where we're putting together a screening methodology where we can actually take a look at targeting multiple different genes and pathways and see which ones are implicated, because I think a lot of the studies that have been done before have been done in animals, and that's not always translatable to humans. So, we focus on the human system and take a look at targets that we can pull out that are relevant for humans.

Fahy: How are you finding these targets? With FISSEQ? (See main Church interview for an explanation of FISSEQ).

Monroe: Yes. FISSEQ will be the downstream final readout.

Dhadwar: We're designing our system so we can target all these different genes at the same time. If we see there's change in our aging metrics then we know we've done something, and say, "Okay, that seemed to reverse this phenotype of aging."

Fahy: You can take a 94-year-old skin cell and make it into an induced pluripotent stem cell (iPS) as well. That has a biological age of 0, and requires only about three to four interventions.

Monroe: Right.

Dhadwar: But that takes a number of weeks to actually reprogram a cell. We're not interested in that. We want something that you can apply right away, one or two days at most, and see if you can actually reverse these aging phenotypes, to actually reverse the aging process without changing the identity of the cell. If you can do that, then you can develop a cocktail that you can apply to the whole body and rejuvenate a number of different tissue types, and it wouldn't be tissue-specific.

Fahy: Yes, for something like your TFAM, that would make sense, because mitochondrial aging will be universal for all cells.

Monroe: Right. Mitochondria are involved in a lot of different disease processes too, so when you think of cancer or neurological disorders, a lot of it arises because the mitochondria aren't working properly.

Fahy: Thank you both for a very exciting look at how rapidly solutions to aging may really be developed.


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