Source: Photo by Clément Falize on Unsplash

Harvard Medical School (HMS) scientists have restored vision in mice by turning back the clock on aged cells in the retina to recapture youthful gene function. In addition to resetting the cells’ aging clock, the investigators demonstrated the reversal of vision loss in animals with a condition mimicking human glaucoma. They say this achievement represents the first successful effort to reverse glaucoma-induced vision loss, rather than merely stem its progression.

The proof-of-concept study represents the first successful attempt to reverse the aging clock in animals through epigenetic reprogramming. If replicated through further preclinical and clinical studies, the same strategy could feasibly allow the development of therapies that promote tissue repair across various organs, not just the retina, and reverse aging and age-related diseases in humans.

“Our study demonstrates that it’s possible to safely reverse the age of complex tissues such as the retina and restore its youthful biological function,” said David Sinclair, PhD, professor of genetics at the Blavatnik Institute at Harvard Medical School, co-director of the Paul F. Glenn Center for Biology of Aging Research at HMS, who is senior author of the team’s published paper in Nature. “If affirmed through further studies, these findings could be transformative for the care of age-related vision diseases like glaucoma and to the fields of biology and medical therapeutics for disease at large,” Sinclair said.

The team report on their studies in a paper titled, “Reprogramming to recover youthful epigenetic information and restore vision.”

Aging is a degenerative process that leads to tissue dysfunction and ultimately death, the researchers wrote. One proposed cause of aging is the accumulation of what they call “epigenetic noise,” which disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity. The researchers’ approach is designed to address this epigenetics-basis theory of aging. The epigenome is effectively a system of turning genes on and off in specific patterns, without altering the basic underlying DNA sequence of the genes. Most cells in the body contain the same genes, but have widely diverse functions, and to achieve this degree of specialization, the cells must read only genes specific to their type, which is the remit of the epigenome.

The epigenetics-related theory of aging postulates that changes to the epigenome over time cause cells to read the wrong genes and malfunction, which then gives rise to diseases of aging. One of the most important changes to the epigenome is DNA methylation, a process by which methyl groups are tacked onto DNA. Patterns of DNA methylation are laid down during embryonic development to produce the various cell types. Over time, youthful patterns of DNA methylation are lost, and genes inside cells that should be switched on get turned off and vice versa, resulting in impaired cellular function.

Some of these DNA methylation changes are predictable and have been used to determine the biologic age of a cell or tissue. “During aging, for reasons that are currently unclear, these patterns change in ways that can be used to calculate DNA methylation age—a representation of biological age that can predict future health and lifespan,” the team noted.

However, whether DNA methylation drives age-related changes inside cells isn’t clear. “Changes to DNA methylation patterns over time form the basis of aging clocks, but whether older individuals retain the information needed to restore these patterns—and, if so, whether this could improve tissue function—is not known,” the scientists pointed out.

For their newly reported study, the researchers hypothesized that if DNA methylation does, indeed, control aging, then erasing some of its footprints might reverse the age of cells inside living organisms and restore them to their earlier, more youthful state. “Having previously found evidence for epigenetic noise as an underlying cause of aging, we wondered whether mammalian cells might retain a faithful copy of epigenetic information from earlier in life that could serve as instructions to reverse aging,” they commented. Past work had achieved this feat in cells grown in laboratory dishes but fell short of demonstrating the effect in living organisms.

Lead study author, Yuancheng Lu, research fellow in genetics at HMS and a former doctoral student in Sinclair’s lab, developed a gene therapy that could safely reverse the age of cells in a living animal. Lu’s work builds on the Nobel Prize winning discovery of Shinya Yamanaka, who identified the four transcription factors, Oct4, Sox2, Klf4, c-Myc, that could erase epigenetics markers on cells and return these cells to their primitive embryonic state from which they can develop into any other type of cell.

Subsequent studies had highlighted two important setbacks, however. First, when used in adult mice, the four Yamanaka factors could also induce tumor growth, rendering the approach unsafe. Second, the factors could reset the cellular state to the most primitive cell state, thus completely erasing a cell’s identity. Lu and colleagues circumvented these hurdles by slightly modifying the approach. They dropped the gene c-Myc and delivered only the remaining three Yamanaka genes, Oct4, Sox2 and Klf4 (collectively known as OSK). The modified approach successfully reversed cellular aging without fueling tumor growth or losing their identity.

To test whether the regenerative capacity of young animals could be achieved in adult mice, the researchers delivered the modified three-gene combination via an adeno-associated virus (AAV) into retinal ganglion cells of adult mice with optic nerve injury. They targeted cells in the central nervous system because it is the first part of body affected by aging. After birth, the ability of the central nervous system to regenerate declines rapidly. Lu and Sinclair partnered with Zhigang He, PhD, HMS professor of neurology and of ophthalmology at Boston Children’s Hospital, who studies optic nerve and spinal cord neuroregeneration.

They found that the treatment had multiple beneficial effects on the eye. It promoted nerve regeneration following optic nerve injury in mice with damaged optic nerves, resulting in a two-fold increase in the number of surviving retinal ganglion cells after the injury, and five-fold increase in nerve regrowth.

“At the beginning of this project, many of our colleagues said our approach would fail or would be too dangerous to ever be used,” said Lu. “Our results suggest this method is safe and could potentially revolutionize the treatment of the eye and many other organs affected by aging.”

Following the encouraging findings in mice with optic nerve injuries, the team partnered with colleagues at Schepens Eye Research Institute of Massachusetts Eye and Ear Bruce Ksander, HMS associate professor of ophthalmology, and Meredith Gregory-Ksander, HMS assistant professor of ophthalmology. They planned two sets of experiments, one to test whether the three-gene cocktail could restore vision loss due to glaucoma, and another to see whether the approach could reverse vision loss stemming from normal aging.

In a mouse model of glaucoma, the treatment led to increased nerve cell electrical activity and a notable increase in visual acuity, as measured by the animals’ ability to see moving vertical lines on a screen. Remarkably, it did so after the glaucoma-induced vision loss had already occurred. “To our knowledge, this is the first example of vision-loss reversal after glaucomatous injury has occurred; previous attempts have focused on neuroprotection delivered at an early stage to prevent further disease progression,” the authors wrote.

Ksander further commented, “Regaining visual function after the injury occurred has rarely been demonstrated by scientists, This new approach, which successfully reverses multiple causes of vision loss in mice without the need for a retinal transplant, represents a new treatment modality in regenerative medicine.”

The treatment worked similarly well in elderly, 12-month-old mice with diminishing vision due to normal aging. Following treatment of the elderly mice, the gene expression patterns and electrical signals of the optic nerve cells were similar to young mice, and vision was restored. When the researchers analyzed molecular changes in treated cells, they found reversed patterns of DNA methylation—an observation suggesting that DNA methylation is not a mere marker or a bystander in the aging process, but rather an active agent driving it. “What this tells us is the clock doesn’t just represent time—it is time,” said Sinclair. “If you wind the hands of the clock back, time also goes backward.”

“Using the eye as a model system, we present evidence that the ectopic expression of OSK transcription factors safely induces in vivo epigenetic restoration of aged CNS neurons, without causing a loss of cell identity or pluripotency,” the researchers wrote. “Instead, OSK promotes a youthful epigenetic signature and gene-expression pattern that causes the neurons to function as though they were young again.”

Encouragingly, for their reported study, a one-year, whole-body treatment of mice with the three-gene approach showed no negative side effects. The researchers said that if their findings are confirmed in further animal work, they could initiate clinical trials within two years to test the efficacy of the approach in people with glaucoma. Thus far, the findings are encouraging, they noted. “These data indicate that mammalian tissues retain a record of youthful epigenetic information—encoded in part by DNA methylation—that can be accessed to improve tissue function and promote regeneration in vivo.”

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