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  1. Go China! NATURE http://www.nature.com/news/chinese-scientists-to-pioneer-first-human-crispr-trial-1.20302?WT.mc_id=TWT_NatureNews Gene-editing technique to treat lung cancer is due to be tested in people in August. David Cyranoski 21 July 2016 Genes in immune cells will be edited in an effort to turbocharge their attack on tumours. Chinese scientists are on the verge of being first in the world to inject people with cells modified using the CRISPR–Cas9 gene-editing technique. A team led by Lu You, an oncologist at Sichuan University’s West China Hospital in Chengdu, plans to start testing such cells in people with lung cancer next month. The clinical trial received ethical approval from the hospital's review board on 6 July. “It’s an exciting step forward,” says Carl June, a clinical researcher in immunotherapy at the University of Pennsylvania in Philadelphia. First CRISPR clinical trial gets green light from US panel There have been a number of human clinical trials using an alternative gene-editing technique, including one led by June, that have helped patients combat HIV. June is also a scientific adviser on a planned US trial that would also use CRISPR–Cas9-modified cells for the treatment of cancer. Last month, an advisory panel of the US National Institutes of Health (NIH) approved that project. But the trial also requires a green light from the US Food and Drug Administration (FDA) and a university review board. The US researchers have said they could start their clinical trial by the end of this year. Ineffective chemo The Chinese trial will enrol patients who have metastatic non-small cell lung cancer and for whom chemotherapy, radiation therapy and other treatments have failed. “Treatment options are very limited,” says Lu. “This technique is of great promise in bringing benefits to patients, especially the cancer patients whom we treat every day.” Leukaemia success heralds wave of gene-editing therapies Lu’s team will extract immune cells called T cells from the blood of the enrolled patients, and then use CRISPR–Cas9 technology — which pairs a molecular guide able to identify specific genetic sequences on a chromosome with an enzyme that can snip the chromosome at that spot — to knock out a gene in the cells. The gene encodes a protein called PD-1 that normally acts as a check on the cell’s capacity to launch an immune response, to prevent it from attacking healthy cells. The gene-edited cells will then be multiplied in the lab and re-introduced into the patient’s bloodstream. The engineered cells will circulate and, the team hopes, home in on the cancer, says Lu. The planned US trial similarly intends to knock out the gene for PD-1, and it will also knock out a second gene and insert a third before the cells are re-introduced into the patient. Last year, the FDA approved for use against lung cancer two antibody-based therapies that block PD-1. But it is difficult to predict for any given patient to what extent these antibodies will block PD-1 and activate the immune response. Gene-editing research in human embryos gains momentum By contrast, knocking out the gene blocks PD-1 with greater certainty, while multiplying the cells increases the chance of a response. “It will be much more powerful than the antibodies," says Timothy Chan, who does clinical research in immunotherapy at Memorial Sloan Kettering Cancer Center in New York City. Validated cells It is well known that CRISPR can result in gene edits at the wrong place in the genome, with potentially harmful effects. Chengdu MedGenCell, a biotechnology company and a collaborator on the trial, will validate the cells to ensure that the correct genes are knocked out before the cells are re-introduced into the patients, says oncologist Lei Deng of West China Hospital, who is a member of Lu’s team. Because the technique targets T cells, which are involved in various kinds of immune responses, in a non-specific way, Chan worries that the approach might induce an excessive autoimmune response in which the cells would start attacking the gut, or adrenaline glands or other normal tissue. “All the T cells — everything will be active. That will be a concern,” says Chan. The unsung heroes of CRISPR He suggests, instead, that the team take T cells from the site of the tumour, because they would already be specialized for attacking cancer. But Deng says that the lung-cancer tumours targeted by their trial are not easily accessible. He also says that the team is reassured by the FDA-approved antibody therapies, which did not show a high rate of autoimmune response. The phase I trial is designed foremost to test whether the approach is safe. It will examine the effects of three different dosage regimens on ten people, and, Deng says, the team plans to proceed slowly, increasing the dosage gradually and starting with just one patient, who will be monitored closely for side effects. But the researchers will also closely watch markers in the blood that would indicate that the treatment is working. Fast reputation Lu says that the review process, which took half a year, required that the team invest a lot of time and human resources, including close communication with the hospital’s internal review board (IRB). “There was a lot of back and forth,” he says. The NIH’s approval of the other CRISPR trial “strengthened ours and our IRB’s confidence in this study”, he adds. China has had a reputation for moving fast — sometimes too fast — with CRISPR, says Tetsuya Ishii, a bioethicist at Hokkaido University in Sapporo, Japan. Bioethics in China: No wild east According to Lu, his team was able to move fast because they are experienced with clinical trials of cancer treatments. June is not surprised that a Chinese group would jump out in front on a trial such as this: “China places a high priority on biomedical research,” he says. Ishii notes that if the clinical trial begins as planned, it would be the latest in a series of firsts for China in the field of CRISPR gene editing, including the first CRISPR-edited human embryos, and the first CRISPR-edited monkeys. “When it comes to gene editing, China goes first,” says Ishii. “I hope we are the first," says Lu. "And more importantly, I hope we can get positive data from the trial.” Nature doi:10.1038/nature.2016.20302
  2. http://www.lifeextension.com/Lpages/2016/CRISPR/index Human Age Reversal at Harvard University 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 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? 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 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! References Available at: https: //www.washingtonpost.com/news/achenblog/wp/2015/12/02/professor-george-church-says-he-can-reverse-the-aging-process/. Accessed April 12, 2016. Available at: http: //today.duke.edu/2015/12/crisprmousedmd. Accessed April 12, 2016. Available at: http: //www.sciencemag.org/topic/2015-breakthrough-year. Accessed April 12, 2016. Available at: http: //www.dddmag.com/news/2016/01/google-backed-gene-editing-startup-files-100m-ipo. Accessed April 12, 2016. Studer L, Vera E, Cornacchia D. Programming and reprogramming cellular age in the era of induced pluripotency. Cell Stem Cell. 2015;16(6): 591-600. Gomes AP, Price NL, Ling AJ, et al. Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging. Cell. 2013;155(7): 1624-38. Poggioli T, Vujic A, Yang P, et al. Circulating growth differentiation factor 11/8 levels decline with age. Circ Res. 2016;118(1): 29-37. Loffredo FS, Steinhauser ML, Jay SM, et al. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 2013;153: 828-39. Sinha M, Jang YC, Oh J, et al. Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science. 2014;344(6184): 649-52. Katsimpardi L, Litterman NK, Schein PA, et al. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science. 2014;344(6184): 630-4. Zhang G, Li J, Purkayastha S, et al. Hypothalamic programming of systemic ageing involving IKK-beta, NF-kappaB and GnRH. Nature. 2013;497(7448): 211-6. Jaskelioff M, Muller FL, Paik JH, et al. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. Nature. 2011;469(7328): 102-6. DePinho RA. The age of cancer. Nature. 2000;408(6809): 248-54. Peters MJ, Joehanes R, Pilling LC, et al. The transcriptional landscape of age in human peripheral blood. Nat Commun. 2015;6: 8570. Horvath S. DNA methylation age of human tissues and cell types. Genome Biol. 2013;14(10): R115. Hannum G, Guinney J, Zhao L, et al. Genome-wide methylation profiles reveal quantitative views of human aging rates. Mol Cell. 2013;49(2): 359-67. Kogan V, Molodtsov I, Menshikov LI, et al. Stability analysis of a model gene network links aging, stress resistance, and negligible senescence. Sci Rep. 2015;5: 13589. Lee JH, Daugharthy ER, Scheiman J, et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc. 2015;10(3): 442-58. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121): 823-6. Sheridan C. First CRISPR-Cas patent opens race to stake out intellectual property. Nat Biotechnol. 2014;32(7): 599-601.
  3. Sthira

    On pursuing Biogerontology

    Your thoughts, if, say, hypothetically you were thinking of entering the fray. I copy and paste from: http://www.senescence.info/biogerontology_career.html How to Become a Biogerontologist senescence.info logo Biogerontologists study the biological process of aging at different levels and using different techniques and models. If you would like to do research on the biology of aging and/or you are a student thinking about pursuing a career in biogerontology then this brief essay is for you. Keywords: age-related diseases, gerontology, gerontologists, jobs By and large, biogerontologists work at research institutions, typically universities and laboratories, though some also work in the biotechnology industry--and a few companies research aging. The vast majority of biogerontologists have a PhD (or sometimes an MD or both), so if you want to become a biogerontologist you should be prepared to go to graduate and/or medical school. While it is possible to study aging in a private company or as a staff member of a research institution, the majority of influential biogerontologists have their own research group, like mine, at a research institution. Again, you can certainly contribute to research on aging in a variety of ways and even without making of it your main job, yet if you are serious about becoming a biogerontologist and doing independent research at the highest level then this usually implies developing an academic career. If you have an entrepreneurial spirit you could create, or help build up, a biotech company with some focus on aging. You could then do research, usually with translation to humans as a shorter-term aim than in academia, that has commercial value. Although there are a few people working on aging who followed this path, they are a minority and I know very little about entrepreneurship so cannot offer much advice on this--but wish you good luck. As such, this essay focuses on academia. How to develop a career in science is the subject of another essay of mine. Briefly, an academic career is highly competitive and usually entails having good grades in high school (in particular in science classes), getting a bachelor's degree with honors and later a doctoral degree (and maybe a master's degree in between, though I normally do not recommend it as top students can often enter a doctoral program without a masters), obtaining strong recommendation letters from advisors, and eventually developing a publication record, securing grants, and doing some teaching. If you are a student, you should have a counselor at your institution that can guide you through the process and there are also many resources on pursuing an academic career on the Internet. One major doubt of students is which topics they should study to prepare themselves for a career in biogerontology. Because aging is a biological process I would suggest that you include biology courses in your education. With the sequencing of the human genome and recent progress in the genetics of aging and longevity, I would also recommend some knowledge of genetics. Nevertheless, do not overestimate the importance of choosing the right courses and university. It does not make such a big difference because many different techniques and skills can be employed to study aging. There are physicists, physicians, engineers, biologists, geneticists, computer scientists, mathematicians, and many other different professionals studying aging right now. Therefore, my advice is for you to learn different skills, understand the science of aging, and focus on the area you find more exciting or more adequate to your personal situation. (As a side note, I would also recommend you develop good communication skills, both written and oral, as these are crucial not only in academia but in many other careers as well.) In the end, remember that who you are is more important than what you learn. To quote Einstein: "Creativity is more important than knowledge." Even though my opinion might be biased, I definitely think my essays on the biology of aging are an excellent introduction to anyone wishing to pursue a career in biogerontology. A briefer overview of gerontology is available as one of my papers (de Magalhaes, 2011). Nonetheless, I also suggest you take a look at my book recommendations since there may be other sources that better fit your personal taste. Lastly, there are occasional intensive courses on the biology of aging, such as the Molecular Biology of Aging course in Woods Hole, MA, USA. As an undergraduate, I would recommend you gain some research experience. This might also help you decide whether doing research is the career for you. For example, you can do an internship in a research group, like ours, and often your mentor or counselor will help you arrange this. I should note that I am always glad to help students aspiring to develop a career in aging research so if you wish to visit our lab or even spend some time here to see what we do just let me know. Once you become more familiar with research in general you will need to start reading academic papers. The main bibliographical database in the biomedical sciences is PubMed and you will need to become familiar with it at some point in your career, possibly in high school or the latest in college. At some point before going to graduate school, I would advise students to start identifying those researchers in the field whose work they most admire and sub-fields of particular interest. This can be done through publications, though often it is difficult for beginners to make sense of the massive archive of publications. Therefore, I would also advise you to look at the list I maintain of researchers working on aging, which includes links to the researchers' websites (if available) and links to PubMed that allow you to more quickly find relevant publications by each of the researchers. Similar to the point made above about how there is no right topic to study, there is no right school or even degree. Assuming your priority is research on aging then having a PhD has advantages over having an MD since it is difficult to balance research and clinical work, but more often than not this is a personal choice and many people enjoy clinical work. Regarding the choice of countries, this is certainly influenced by the mobility of each individual but a few points may be worth considering. The US and European systems are different in regard to graduate and medical schools. For example, graduate students in the US usually take longer to receive their PhDs. Mostly because of this, and please have in mind that I am an European, I normally would not recommend for an European to get his/her PhD in the US, but like many other suggestions in this essay this is often a personal decision. It is also possible to get a PhD from an European institution but do part or even most of the work in the US. Likewise, many people carry out most of their doctoral work in Europe or in the US but then receive their PhDs from institutions in other countries, often their native countries. Overall, working on aging has its challenges, such as the lack of adequate models of human aging and a lack of funding when compared to other biomedical disciplines. Salary-wise, in fact, working in academia is not the best career choice. Scientists usually do research because they love it, not because they want to become millionaires, though some scientists are also associated with industry which brings in extra income. Working in academia does have its advantages, such as schedule flexibility and creative freedom. Besides, since the field of aging is still largely mysterious with many unanswered questions, bright young minds have an extraordinary opportunity to make important contributions to science by studying aging. I hope you will be one of those minds. Please feel free to contact me if you have questions not covered in this essay or need any advice. Up to the Visitor's Resources Back to senescence.info Thank you for visiting my website. Please feel free to contact me if you have any questions, ideas, comments or suggestions. Copyright © 2007, 2008, 2012 by João Pedro de Magalhães. All rights reserved.
  4. The blog Fight Aging! (which I highly recommend), has a nice, comprehensive year-in-review article on the progress made in 2015 towards defeating aging. It focuses a lot on progress made by the SENS Research Foundation projects, but also discusses commercial efforts like Human Longevity Inc and Calico, and the government sponsored longevity trial of metformin, along with research progress on clearing amyloid plaques for Alzheimer's disease, CRISPR technology for gene editing, cryonics, printing new organs, and more! Well worth a read for anyone interested in lifespan extension. P.S. Happy New Year! --Dean
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