Epigenic Rejuvenation

Aug 8, 2019
Has this scientist finally found the fountain of youth?
Editing the epigenome, which turns our genes on and off, could be the “elixir of life.”
by Erika Hayasaki

The black mouse on the screen sprawls on its belly, back hunched, blinking but otherwise motionless. Its organs are failing. It appears to be days away from death. It has progeria, a disease of accelerated aging, caused by a genetic mutation. It is only three months old.

I am in the laboratory of Juan Carlos Izpisúa Belmonte, a Spaniard who works at the Gene Expression Laboratory at San Diego’s Salk Institute for Biological Studies, and who next shows me something hard to believe. It’s the same mouse, lively and active, after being treated with an age-reversal mixture. “It completely rejuvenates,” Izpisúa Belmonte tells me with a mischievous grin. “If you look inside, obviously, all the organs, all the cells are younger.”

Izpisúa Belmonte, a shrewd and soft-spoken scientist, has access to an inconceivable power. These mice, it seems, have sipped from a fountain of youth. Izpisúa Belmonte can rejuvenate aging, dying animals. He can rewind time. But just as quickly as he blows my mind, he puts a damper on the excitement. So potent was the rejuvenating treatment used on the mice that they either died after three or four days from cell malfunction or developed tumors that killed them later. An overdose of youth, you could call it.

The powerful tool that the researchers applied to the mouse is called “reprogramming.” It’s a way to reset the body’s so-called epigenetic marks: chemical switches in a cell that determine which of its genes are turned on and which are off. Er3ase these marks and a cell can forget if it was ever a skin or a bone cell, and revert to a much more primitive, embryonic state. The technique is frequently used by laboratories to manufacture stem cells. But Izpisúa Belmonte is in a vanguard of scientists who want to apply reprogramming to whole animals and, if they can control it precisely, to human bodies.

Izpisúa Belmonte believes epigenetic reprogramming may prove to be an “elixir of life” that will extend human life span significantly. Life expectancy has increased more than twofold in the developed world over the past two centuries. Thanks to childhood vaccines, seat belts, and so on, more people than ever reach natural old age. But there is a limit to how long anyone lives, which Izpisúa Belmonte says is because our bodies wear down through inevitable decay and deterioration. “Aging,” he writes, “is nothing other than molecular aberrations that occur at the cellular level.” It is, he says, a war with entropy that no individual has ever won.

“I think the kid that will be living to 130 is already with us. He has already been born. I’m convinced.”

But each generation brings new possibilities, as the epigenome gets reset during reproduction when a new embryo is formed. Cloning takes advantage of reprogramming, too: a calf cloned from an adult bull contains the same DNA as the parent, just refreshed. In both cases, the offspring is born without the accumulated “aberrations” that Izpisúa Belmonte refers to.

What Izpisúa Belmonte is proposing is to go one step better still, and reverse aging-related aberrations without having to create a new individual. Among these are changes to our epigenetic marks—chemical groups called histones and methylation marks, which wrap around a cell’s DNA and function as on/off switches for genes. The accumulation of these changes causes the cells to function less efficiently as we get older, and some scientists, Izpisúa Belmonte included, think they could be part of why we age in the first place. If so, then reversing these epigenetic changes through reprogramming may enable us to turn back aging itself.

Izpisúa Belmonte cautions that epigenetic tweaks won’t “make you live forever,” but they might delay your expiration date. As he sees it, there is no reason to think we cannot extend human life span by another 30 to 50 years, at least. “I think the kid that will be living to 130 is already with us,” Izpisúa Belmonte says. “He has already been born. I’m convinced.”

Youth factors

The treatment Izpisúa Belmonte gave his mice is based on a Nobel-winning discovery by the Japanese stem-cell scientist Shinya Yamanaka. Starting in 2006, Yamanaka demonstrated how adding just four proteins to human adult cells could reprogram them so that they look and act like those in a newly formed embryo. These proteins, called the Yamanaka factors, function by wiping clean the epigenetic marks in a cell, giving it a fresh start.

“He went backwards in time,” Izpisúa Belmonte says. All the methylation marks, those epigenetic switches, “are erased,” he adds. “Then you’re starting life again.” Even skin cells from centenarians, scientists have found, can be rewound to a primitive, youthful state. The artificially reprogrammed cells are called induced pluripotent stem cells, or IPSCs. Like the stem cells in embryos, they can then turn into any kind of body cell—skin, bone, muscle, and so on—if given the right chemical signals.

To many scientists, Yamanaka’s discovery was promising mainly as a way to manufacture replacement tissue for use in new types of transplant treatments. In Japan, researchers began an effort to reprogram cells from a Japanese woman in her 80s with a blinding disease, macular degeneration. They were able to take a sample of her cells, return them to an embryonic state with Yamanaka’s factors, and then direct them to become retinal cells. In 2014, the woman became the first person to receive a transplant of such lab-made tissue. It didn’t make her vision sharper, but she did report it as being “brighter,” and it stopped deteriorating.

Before then, though, researchers at the Spanish National Cancer Research Centre had already taken the technology in a new direction when they studied mice whose genomes harbored extra copies of the Yamanaka factors. Turning these on, they demonstrated that cell reprogramming could actually occur inside an adult animal body, not only in a laboratory dish.

The experiment suggested an entirely new form of medicine. You could potentially rejuvenate a person’s entire body. But it also underscored the dangers. Clear away too many of the methylation marks and other footprints of the epigenome and “your cells basically lose their identity,” says Pradeep Reddy, a staff researcher at Salk who worked on these experiments with Izpisúa Belmonte. “You are erasing their memory.” These cellular blank slates can grow into a mature, functioning cell, or into one that never develops the ability to perform its designated task. It can also become a cancer cell.

That’s why the mice I saw in Izpisúa Belmonte’s lab were prone to sprouting tumors. It proved that cellular reprogramming had indeed occurred inside their bodies, but the results were usually fatal.

Izpisúa Belmonte believed there might be a way to give mice a less lethal dose of reprogramming. He was inspired by salamanders, which can regrow an arm or tail. Researchers have yet to determine exactly how amphibians do this, but one theory is that it happens through a process of epigenetic resetting similar to what the Yamanaka factors achieve, though more limited in scope. With salamanders, their cells “just go back a little bit” in time, Izpisúa Belmonte says.

Could the same thing be done to an entire animal? Could it be rejuvenated just enough?

In 2016, the team devised a way to partially rewind the cells in mice with progeria. They genetically modified the mice to produce the Yamanaka factors in their bodies, just as the Spanish researchers had done; but this time, the mice would produce those factors only when given an antibiotic, doxycycline.

In Izpisúa Belmonte’s lab, some mice were allowed to drink water containing doxycycline continuously. In another experiment, others got it just for two days out of every seven. “When you give them … doxycycline, expression of the genes starts,” explains Reddy. “The moment you remove it, the expression of the genes stops. You can easily turn it on or off.”

The mice that drank the most, like the one Izpisúa Belmonte showed me, quickly died. But the mice that drank a limited dose did not develop tumors. Instead, they became more physically robust, their kidneys and spleens worked better, and their hearts pumped harder.

In all, the treated mice also lived 30% longer than their littermates. “That was the benefit,” Izpisúa Belmonte says. “We don’t kill the mouse. We don’t generate tumors, but we have our rejuvenation.”

Fountain of youth

When Izpisúa Belmonte published his report in the journal Cell, describing the rejuvenated mice, it seemed to some as if Ponce de Leon had finally spotted the fountain of youth. “I think Izpisúa Belmonte’s paper woke a lot of people up,” says Michael West, CEO of AgeX, which is pursing similar aging reversal technology. “All of a sudden all of the leaders in aging research are like, ‘Oh, my gosh, this could work in the human body.’”

To West, the technology offers the prospect that humans, like salamanders, could regenerate tissues or damaged organs. “Humans have that ability too, when we are first forming,” he says. “So if we can reawaken those pathways ... wow!”

To others, however, the evidence for rejuvenation is plainly in its infancy. Jan Vijg, chair of the genetics department at the Albert Einstein College of Medicine in New York City, says aging consists of “hundreds of different processes” to which simple solutions are unlikely. Theoretically, he believes, science can “create processes that are so powerful they could override all of the other ones.” But he adds, “We don’t know that right now.”

An even broader doubt is whether the epigenetic changes that Izpisúa Belmonte is reversing in his lab are really the cause of aging or just a sign of it—the equivalent of wrinkles in aging skin. If so, Izpisúa Belmonte’s treatment might be like smoothing out wrinkles, a purely cosmetic effect. “We have no way of knowing, and there is really no evidence, that says the DNA methylation [is] causing these cells to age,” says John Greally, another professor at Einstein. The notion that “if I change those DNA methylations, I will be influencing aging,” he says, “has red flags all over it.”

One other fundamental question hangs over Izpisúa Belmonte’s findings: while he succeeded in rejuvenating mice with progeria, he hasn’t done it in normal aged animals. Progeria is an illness due to a single DNA mutation. Natural aging is much more complex, says Vittorio Sebastiano, an assistant professor at the Stanford Institute for Stem Cell Biology and Regenerative Medicine. Would the rejuvenation technique work in naturally aged animals and in human cells? He says Izpisúa Belmonte’s research so far leaves that crucial question unanswered.

Izpisúa Belmonte’s team is working to answer it. Experiments to rejuvenate normal mice are under way. But because normal mice live as long as two and a half years, whereas those with progeria live three months, the evidence is taking longer to gather. “And if we have to modify any experimental condition,” Reddy says, “then the whole cycle will have to be repeated.”

Editing age

Wholesale rejuvenation, then, is still far off, if it will ever come at all. But more limited versions of it, targeted to certain diseases of aging, might be available within a few years.

If the Yamanaka factors are like a scattergun that wipes out all the epigenetic marks associated with aging, the techniques now being developed at Salk and in other labs are more like sniper rifles. The goal is to allow researchers to switch off a specific gene that causes a disease, or switch on another gene that can alleviate it.

Hsin-Kai Liao and Fumiyuki Hatanaka spent four years in Izpisúa Belmonte’s lab adapting CRISPR-Cas9, the famed DNA “editing” system, to instead act as a volume control knob. Whereas the original CRISPR lets researchers eliminate an unwanted gene, the adapted tool allows them to leave the genetic code untouched but determine whether a gene is turned on or off.

The lab has tested this tool on mice with muscular dystrophy, which lack a gene that’s crucial in maintaining muscle. Using the epigenome editor, the researchers cranked up the output of another gene that can play a substitute role. The mice they treated did better on grip tests, and their muscles “had become much larger,” Liao remembers.

Another result of this kind came from beyond the Salk campus, at the University of California, Irvine. Researcher Marcelo Wood claims that activating a single gene in old mice improves their memory in a test involving moving objects. “We restored long-term memory function in those animals,” says Wood, who published the results in Nature Communications. After a single epigenetic block is removed, says Wood, “the genes for memory—they all fire. Now that animal perfectly encodes that information straight into long-term memory.”

“I think turning back the clock is an appropriate way to explain it.”

Similarly, researchers at Duke University have developed an epigenetic editing technique (not yet tested on animals) to turn down the volume on a gene implicated in Parkinson’s disease. Another Duke team brought down the levels of cholesterol in mice by turning off a gene that regulates it. Izpisúa Belmonte’s lab itself, as well as experimenting with muscular dystrophy, has worked on rolling back the symptoms of diabetes, kidney disease, and the loss of bone cartilage, all using similar methods.

The first human tests of these techniques are likely to happen in the next few years. Two companies pursuing the technology are AgeX and Turn Biotechnologies, a startup cofounded by Sebastiano from Stanford. AgeX, says West, its CEO, is looking to target heart tissues, while Turn, according to Sebastiano, will begin by seeking regulatory clearance to test treatments for osteoarthritis and aging-related muscle loss.

Meanwhile GenuCure, a biotech company founded by Ilir Dubova, a former researcher at Salk, is raising funds to pursue an idea for rejuvenating cartilage. The company has a “cocktail,” Dubova says, that will be injected into the knee capsule of people with osteoarthritis, perhaps once or twice a year. Such a treatment could take the place of expensive knee replacement surgeries.

“After injection, these … genes that were silenced due to aging would be turned on, thanks to our witchcraft, and start the rejuvenation process of the tissue,” Dubova says. “I think turning back the clock is an appropriate way to explain it.”


Turning back time: Salk scientists reverse signs of aging
New technique rejuvenated organs and helped animals live longer

LA JOLLA—Graying hair, crow’s feet, an injury that’s taking longer to heal than when we were 20—faced with the unmistakable signs of aging, most of us have had a least one fantasy of turning back time. Now, scientists at the Salk Institute have found that intermittent expression of genes normally associated with an embryonic state can reverse the hallmarks of old age.

This approach, which not only prompted human skin cells in a dish to look and behave young again, also resulted in the rejuvenation of mice with a premature aging disease, countering signs of aging and increasing the animals’ lifespan by 30 percent. The early-stage work provides insight both into the cellular drivers of aging and possible therapeutic approaches for improving human health and longevity.

“Our study shows that aging may not have to proceed in one single direction,” says Juan Carlos Izpisua Belmonte, a professor in Salk’s Gene Expression Laboratory and senior author of the paper appearing in the December 15, 2016, issue of Cell. “It has plasticity and, with careful modulation, aging might be reversed.”

As people in modern societies live longer, their risk of developing age-related diseases goes up. In fact, data shows that the biggest risk factor for heart disease, cancer and neurodegenerative disorders is simply age. One clue to halting or reversing aging lies in the study of cellular reprogramming, a process in which the expression of four genes known as the Yamanaka factors allows scientists to convert any cell into induced pluripotent stem cells (iPSCs). Like embryonic stem calls, iPSCs are capable of dividing indefinitely and becoming any cell type present in our body.

“What we and other stem-cell labs have observed is that when you induce cellular reprogramming, cells look younger,” says Alejandro Ocampo, a research associate and first author of the paper. “The next question was whether we could induce this rejuvenation process in a live animal.”

While cellular rejuvenation certainly sounds desirable, a process that works for laboratory cells is not necessarily a good idea for an entire organism. For one thing, although rapid cell division is critical in growing embryos, in adults such growth is one of the hallmarks of cancer. For another, having large numbers of cells revert back to embryonic status in an adult could result in organ failure, ultimately leading to death. For these reasons, the Salk team wondered whether they could avoid cancer and improve aging characteristics by inducing the Yamanaka factors for a short period of time.

To find out, the team turned to a rare genetic disease called progeria. Both mice and humans with progeria show many signs of aging including DNA damage, organ dysfunction and dramatically shortened lifespan. Moreover, the chemical marks on DNA responsible for the regulation of genes and protection of our genome, known as epigenetic marks, are prematurely dysregulated in progeria mice and humans. Importantly, epigenetic marks are modified during cellular reprogramming.

Using skin cells from mice with progeria, the team induced the Yamanaka factors for a short duration. When they examined the cells using standard laboratory methods, the cells showed reversal of multiple aging hallmarks without losing their skin-cell identity.

“In other studies scientists have completely reprogrammed cells all the way back to a stem-cell-like state,” says co-first author Pradeep Reddy, also a Salk research associate. “But we show, for the first time, that by expressing these factors for a short duration you can maintain the cell’s identity while reversing age-associated hallmarks.”

Encouraged by this result, the team used the same short reprogramming method during cyclic periods in live mice with progeria. The results were striking: Compared to untreated mice, the reprogrammed mice looked younger; their cardiovascular and other organ function improved and—most surprising of all—they lived 30 percent longer, yet did not develop cancer. On a cellular level, the animals showed the recovery of molecular aging hallmarks that are affected not only in progeria, but also in normal aging.

“This work shows that epigenetic changes are at least partially driving aging,” says co-first author Paloma Martinez-Redondo, another Salk research associate. “It gives us exciting insights into which pathways could be targeted to delay cellular aging.”

Lastly, the Salk scientists turned their efforts to normal, aged mice. In these animals, the cyclic induction of the Yamanaka factors led to improvement in the regeneration capacity of pancreas and muscle. In this case, injured pancreas and muscle healed faster in aged mice that were reprogrammed, indicating a clear improvement in the quality of life by cellular reprogramming.

“Obviously, mice are not humans and we know it will be much more complex to rejuvenate a person,” says Izpisua Belmonte. “But this study shows that aging is a very dynamic and plastic process, and therefore will be more amenable to therapeutic interventions than what we previously thought.”

The Salk researchers believe that induction of epigenetic changes via chemicals or small molecules may be the most promising approach to achieve rejuvenation in humans. However, they caution that, due to the complexity of aging, these therapies may take up to 10 years to reach clinical trials.

Other authors included: Aida Platero-Luengo, Fumiyuki Hatanaka, Tomoaki Hishida, Mo Li, David Lam, Masakazu Kurita, Ergin Beyret, Toshikazu Araoka, Eric Vazquez-Ferrer, David Donoso, Jose Luis Roman, Jinna Xu and Concepcion Rodriguez of the Salk Institute; Estrella Nuñez Delicado of Universidad Católica San Antonio de Murcia; Gabriel Núñez of the University of Michigan Medical School; Josep Maria Campistol of Hosplital Clinic of Barcelona and Isabel Guillén and Pedro Guillén of Fundación Dr. Pedro Guillén.

The work and the researchers involved were supported in part by a National Institutes of Health Ruth L. Kirschstein National Research Service Award Individual Postdoctoral Fellowship, the Muscular Dystrophy Association, Fundación Alfonso Martin Escudero, the Hewitt Foundation, the Uehara Memorial Foundation, the Nomis Foundation, a JSPS Postdoctoral Fellowship for Research Abroad, the University of California, San Diego, the G. Harold and Leila Y. Mathers Charitable Foundation, The Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002), The Glenn Foundation, Universidad Católica San Antonio de Murcia (UCAM) and Fundación Dr. Pedro Guillén.

Cell, Volume 167, ISSUE 7, P1719-1733.e12, December 15, 2016

In vivo amelioration of age-associated hallmarks by partial reprogramming
Alejandro Ocampo, et al.
Partial reprogramming erases cellular markers of aging in mouse and human cells
Induction of OSKM in progeria mice ameliorates signs of aging and extends lifespan
In vivo reprogramming improves regeneration in 12-month-old wild-type mice

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

For humans living in modern societies, aging is the largest risk factor for most diseases (Partridge, 2014). However, despite decades of effort, the complexity of cellular and organismal aging has limited our understanding of this critical biological process. Epigenetic alterations (e.g., DNA methylation, post-translational modifications of histones, and chromatin remodeling) have recently emerged as one of the most conserved hallmarks of aging (Benayoun et al., 2015, Sen et al., 2016). Interestingly, cellular reprogramming to pluripotency occurs through a stepwise global epigenetic remodeling (Benayoun et al., 2015, Liu et al., 2013b, Polo et al., 2012). The rejuvenation of aging hallmarks has been extensively described during cellular reprogramming to pluripotency in vitro (Lapasset et al., 2011, Liu et al., 2011, Mahmoudi and Brunet, 2012, Rando and Chang, 2012), but the dynamics of this process remain poorly understood. In addition, until now, the amelioration of age-associated phenotypes by cellular reprogramming has not yet been demonstrated at the organismal level.

Our results demonstrate that partial reprogramming by short-term expression of the Yamanaka factors has the capacity to rejuvenate cellular phenotypes of aging in mouse and human cells. Although previous studies have indicated that expression of the Yamanaka factors in vivo can lead to cancer development or teratoma formation (Abad et al., 2013, Ohnishi et al., 2014), here, we demonstrate that tumor formation can be avoided by short-term induction of OSKM. Cyclic induction of OSKM in vivo ameliorated hallmarks of aging and extended the lifespan of a mouse model of premature aging. Additionally, short-term induction of OSKM improved the regenerative capacity of pancreas and muscle following injury in physiologically aged mice. Together, these results show that partial in vivo reprogramming might be used to modulate aging hallmarks and significantly benefit organismal health.

Nature volume 506, pages 304–305 (20 February 2014)
Genetic rejuvenation of old muscle
Mo Li & Juan Carlos Izpisua Belmonte

In advanced age, the stem cells responsible for muscle regeneration switch from reversible quiescence to irreversible senescence. Targeting a driver of senescence revives muscle stem cells and restores regeneration.

One of the telltale signs of advanced ageing is loss of skeletal-muscle mass and strength, a phenomenon known as sarcopenia. Muscle strength is inversely correlated with mortality in old populations1,2, and the decline in strength is attributable to the decreased regenerative capacity of muscle stem cells, called satellite cells, with age. Whether this decline is caused by cell-intrinsic and/or environmental alterations has remained unclear. On page 316 of this issue, Sousa-Victor et al.3 shed light on this debate by uncovering intrinsic aspects of age-related satellite-cell dysfunction that account for the loss of muscle maintenance (homeostasis) and regeneration. This study also provides a potential strategy for satellite-cell rejuvenation that could benefit geriatric individuals and those with progeria, a disorder in which cells age prematurely...

Finally, this study presents yet another addition to the list of potential strategies to improve the regenerative capacity of aged tissue11,14,15. It may be worth considering whether the benefits of transiently reducing tumour-suppressor levels in stem cells outweigh the associated risks, in the context of preventing an age-related decline in regenerative potential. Whether these strategies can be safely implemented in the clinic to maximize human health span deserves thorough investigation in the near future.

Epigenetics Chromatin. 2018; 11: 73.
2018 Dec 20.
doi: 10.1186/s13072-018-0244-7
Age reprogramming and epigenetic rejuvenation
Prim B. Singh, et al.
Age reprogramming represents a novel method for generating patient-specific tissues for transplantation. It bypasses the de-differentiation/redifferentiation cycle that is characteristic of the induced pluripotent stem (iPS) and nuclear transfer-embryonic stem (NT-ES) cell technologies that drive current interest in regenerative medicine. Despite the obvious potential of iPS and NT-ES cell-based therapies, there are several problems that must be overcome before these therapies are safe and routine. As an alternative, age reprogramming aims to rejuvenate the specialized functions of an old cell without de-differentiation; age reprogramming does not require developmental reprogramming through an embryonic stage, unlike the iPS and NT-ES cell-based therapies. Tests of age reprogramming have largely focused on one aspect, the epigenome. Epigenetic rejuvenation has been achieved in vitro in the absence of de-differentiation using iPS cell reprogramming factors. Studies on the dynamics of epigenetic age (eAge) reprogramming have demonstrated that the separation of eAge from developmental reprogramming can be explained largely by their different kinetics. Age reprogramming has also been achieved in vivo and shown to increase lifespan in a premature ageing mouse model. We conclude that age and developmental reprogramming can be disentangled and regulated independently in vitro and in vivo.

Method of making induced pluripotent stem cells
Shinya YAMAKANA, et al.

A method of producing an induced pluripotent stem cell includes introducing into a somatic cell one or more non-viral expression vectors. The vectors include one or more of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and Nanog gene. The somatic cell is then cultured in a medium that supports pluripotent stem cells. At least a portion of the one or more introduced non-viral expression vectors is not substantially integrated in the chromosome...

Technical Problem
It is an object of the present invention to provide a method of producing an iPS cell by reprogramming a somatic cell without using a viral vector such as a retrovirus.

Solution to Problem
The present inventors extensively investigated to solve the problems described above, and found that an iPS cell can be produced by introducing genes that encode reprogramming factors into a somatic cell by means of a non-viral expression vector such as a plasmid vector, and that a safe iPS cell can be obtained from a somatic cell by the method. The present invention has been developed on the basis of these findings.

Accordingly, the present invention provides a method of producing an induced pluripotent stem cell, comprising the step of introducing at least one kind of non-viral expression vector incorporating at least one gene that encodes a reprogramming factor into a somatic cell...



Disclosed herein are homology-independent targeted integration methods of integrating an exogenous DNA sequence into a genome of a non-dividing cell and compositions for such methods. Methods herein comprise contacting the non-dividing cell with a composition comprising a targeting construct comprising the exogenous DNA sequence and a targeting sequence, a complementary strand oligonucleotide homologous to the targeting sequence, and a nuclease, thereby altering the genome of the non-dividing cell.

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