This popular science piece covers some of the high points of recent years in the field of calorie restriction research. It is above average for the type, though it has to be said that the bar has been set low by the media in recent years. This long-standing area of research involves quantifying the benefits to health and longevity produced by consuming fewer calories while still obtaining an optimal level of micronutrients, mapping the cellular mechanisms involved, and a search for ways to recreate some of these effects via calorie restriction mimetic drugs rather than diet.

Thanks to advances in medicine and improvements in healthy living, we benefiting from longer lifespans and also experiencing longer "healthspans". So, what do we need to do to enhance the length and quality of our lives even more? Researchers worldwide are pursuing various ideas, but for some researchers, the answer is a simple change in diet. They believe that the key to a better old age may be to reduce the amount of food on our plates, via an approach called "calorie restriction". This diet goes further than cutting back on fatty foods from time-to-time; it's about making gradual and careful reductions in portion size permanently.

Since a foundational study in 1935 in white rats, a dietary restriction of between 30-50% has been shown to extend lifespan, delaying death from age-related disorders and disease. Of course, what works for a rat or any other laboratory organism might not work for a human. Long-term trials, following humans from early adulthood to death, are a rarity. "I don't see a human study of longevity as something that would be a fundable research programme. Even if you start humans at 40 or 50 years old, you're still looking at potentially 40 or 50 more years of study." That's why, in the late 1980s, two independent long-term trials - one at NIA and the other at the University of Wisconsin - were set up to study calorie restriction and ageing in Rhesus monkeys. Not only do we share 93% of our DNA with these primates, we age in the same way too.

They are far from malnourished or starving. Take Sherman, a 43-year-old monkey from NIA. Since being placed on the CR diet in 1987, aged 16, Sherman hasn't shown any overt signs of hunger that are well characterised in his species. Sherman is the oldest Rhesus monkey ever recorded, nearly 20 years older than the average lifespan for his species in captivity. Even into his 30s he would have been considered an old monkey, but he didn't look or act like one. The same is true, to varying extents, for the rest of his experimental troop at NIA. "We have demonstrated that ageing can be manipulated in primates. It kind of gets glossed over because it's obvious, but conceptually that's hugely important; it means that ageing itself is a reasonable target for clinical intervention and medical treatment."

If ageing can be delayed, in other words, all of the diseases associated with it will follow suit. "Going after each disease one at a time isn't going to significantly extend lifespan for people because they'll die of something else. If you cured all cancers, you wouldn't offset death due to cardiovascular disease, or dementia, or diabetes-associated disorders. Whereas if you go after ageing you can offset the lot in one go."

In the Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy trial, also known as Calerie, over two years, 218 healthy men and women aged between 21 and 50 years were split into two groups. In one, people were allowed to eat as they normally would (ad libitum), while the other ate 25% less (CR). Both had health checks every six months. Unlike in the Rhesus monkey trials, tests over two years can't determine whether CR reduces or delays age-related diseases. There simply isn't enough time for their development. But the Calerie trials tested for the next best thing: the early biological signs of heart disease, cancer, and diabetes. The results after two years were very positive. In the blood of calorie-restricted people, the ratio of "good" cholesterol to "bad" cholesterol had increased, molecules associated with tumour formation - called tumour necrosis factors (TNFs) - were reduced by around 25%, and levels of insulin resistance, a sure sign of diabetes, fell by nearly 40% compared to people who ate their normal diets. Overall, blood pressure was lower.

With less food, is the metabolism forced to be more efficient with what it has? Is there a common molecular switch regulating ageing that is turned on (or off) with fewer calories? Or is there an as of yet unknown mechanism underpinning our lives and deaths? Answers to such questions might be long in coming. "If I cloned 10 of myself and we all worked furiously, I don't think we'd have it solved. The biology is inordinately complicated." It's a worthwhile undertaking - understand how CR works and other treatments could then be used to target that specific part of our biology. Ageing could be treated directly, that is, without the need of calorie restriction.

Link: http://www.bbc.com/future/story/20170601-the-secret-to-a-long-and-healthy-life-eat-less

Aging is an accumulation of molecular damage and its consequences. The greater the level of damage, the greater the dysfunction in organs and the immune system, then the closer the individual comes to the arbitrary dividing line at which that dysfunction becomes a formal, named age-related disease. Further, the more damage, the higher the mortality rate. Given this view of aging, it should be no great surprise to find that the longest lived people have a history of comparatively little age-related disease: the only ways to become extremely old are to either (a) have accumulated damage at a slower rate that everyone else, most likely through lifestyle choices, or (b) bear genetic variants that increase resistance to some forms of damage and consequence. In either case, cell and tissue damage, aging, longevity, and age-related disease are all linked together, facets of the same whole.

In a large cohort of predominantly male community-dwelling elderly veterans, centenarians had a lower incidence of chronic illness than those in their 80s and 90s. The centenarian population is one of the fastest growing in the country, according to the United States Social Security Administration. They are predicted to exceed one million by the close of this century, little is known about why this generation has achieved such longevity. In a recent study, researchers looked primarily at octogenarians, nonagenarians, and centenarians within the Veterans Affairs medical system. The sample that they studied comprised mostly of white males that had fought in World War II. "Additionally, this generation lived through the Great Depression. It is a wonder, considering the hardships they had faced, that they have achieved such longevity."

A key factor that the research team observed in these individuals is that, due to their military background, many had a developed sense of discipline and therefore were keen to make healthy decisions; many did not smoke or drink. The team also offered the hypothesis of compression of morbidity as a potential explanation for the extended health span in an individual's life span. The hypothesis states that the lifetime burden of illness could be reduced if the onset of chronic illness is postponed until very late in life, or in other words "the older you get, the healthier you have been."

Ninety-seven percent of centenarians were male, 88.0% were white, 31.8% were widowed, 87.5% served in World War II, and 63.9% did not have a service-related disability. The incidence rates of chronic illnesses were higher in octogenarians than centenarians (atrial fibrillation, 15.0% vs 0.6%; heart failure, 19.3% vs 0.4%; chronic obstructive pulmonary disease, 17.9% vs 0.6%; hypertension, 29.6% vs 3.0%; end-stage renal disease, 7.2% vs 0.1%; malignancy, 14.1% vs 0.6%; diabetes mellitus, 11.1% vs 0.4%; stroke, 4.6% vs 0.4%) and in nonagenarians than centenarians (atrial fibrillation, 13.2% vs 3.5%; heart failure, 15.8% vs 3.3%; chronic obstructive pulmonary disease, 11.8% vs 3.5%; hypertension, 27.2% vs 12.8%; end-stage renal disease, 11.9% vs 4.5%; malignancy, 8.6% vs 2.3%; diabetes mellitus, 7.5% vs 2.2%; and stroke, 3.5% vs 1.3%).

Link: https://smhs.gwu.edu/news/gw-researcher-finds-centenarians-have-lower-incidence-chronic-illness-contributing-longer

The balance between different types of the immune cells known as macrophages is becoming a stronger theme these days, a line of research that falls somewhere into the broad overlap between regeneration, inflammation, and aging. I've seen quite a number of interesting papers on this topic in the past year, which seems to me a leap in the level of interest shown by the research community of late. While possibly oversimplifying a more complicated reality, we can think of macrophages as having a few different types, or polarizations. The M1 polarization tends towards aggressive destruction of problem cells, the creation of inflammation, and hindrance of regeneration. The M2 polarization tends towards suppression of inflammation and other behaviors that encourage regeneration. The cancer research community would like to be able to adjust macrophage populations towards the M1 type, more willing to destroy cancerous cells, while the regenerative medicine community would like to be able to adjust macrophage populations towards the M2 type to spur enhanced regeneration and tissue maintenance.

It may be that the increased interest in macrophage polarization is a function of the emergence of tools that now allow for cost-effective attempts to shift the balance of macrophage types. The infrastructure of biotechnology is advancing rapidly, and progress spurred by falling costs is a common theme in many parts of the field. Today I'll offer up another example of macrophage polarization research, this time involving microglia, a form of macrophage resident in the central nervous system. Changes in microglia have been shown to be important in any number of age-related neurodegenerative conditions: the immune system declines with age in the brain, just as elsewhere in the body, falling into a dysfunctional and inflammatory state. This affects regeneration and tissue maintenance as is the case for macrophages beyond the brain, but microglia also have additional roles in the correct function of neurons and neural connections, an area of our biochemistry that is still comparatively poorly understood. It is possible to achieve benefits for patients by coercing more microglia into the M2, pro-regenerative polarization? In this open access paper, researchers examine the question in the context of Parkinson's disease.

Targeting Microglial Activation States as a Therapeutic Avenue in Parkinson's Disease

A growing body of evidence suggest that neuroinflammation mediated by microglia, the resident macrophage-like immune cells in the brain, play a contributory role in Parkinson's disease (PD) pathogenesis. In the central nervous system (CNS), the innate immune response is predominantly mediated by microglia and astrocytes. Microglia play a vital role in both physiological and pathological conditions. Microglia appear to be involved in several regulatory processes in the brain that are crucial for tissue development, maintenance of the neural environment and, response to injury and promoting repair. Similar to peripheral macrophages, microglia directly respond to pathogens and maintain cellular homeostasis by purging said pathogens, as well as dead cells and pathological gene products.

Microglia participate in both physiological and pathological conditions. In the former, microglia restore the integrity of the central nervous system and, in the latter, they promote disease progression. Microglia acquire different activation states to modulate these cellular functions. When classically activated, microglia acquire the M1 phenotype, characterized by pro-inflammatory and pro-killing functions that serve as the first line of defense. The alternative M2 microglial activation state is involved in various events including immunoregulation, inflammation dampening, and repair and injury resolution.

Upon activation to the M1 phenotype, microglia elaborate pro-inflammatory cytokines and neurotoxic molecules promoting inflammation and cytotoxic responses. In contrast, when adopting the M2 phenotype microglia secrete anti-inflammatory gene products and trophic factors that promote repair, regeneration, and restore homeostasis. Relatively little is known about the different microglial activation states in PD, and the distribution of microglial M1/M2 phenotypes depends on the stage and severity of the disease. Understanding stage-specific switching of microglial phenotypes and the capacity to manipulate these transitions within appropriate time windows might be beneficial for PD therapy. The transition from the M1 pro-inflammatory state to the regulatory or anti-inflammatory M2 phenotype is thought to assist improved functional outcomes and restore homeostasis. The induction of M1 phenotype is a relatively standard response during injury. For peripheral immune cells it is thought that M1 polarization is terminal and the cells die during the inflammatory response. Although a shift from M1 to the M2 phenotype is considered rare for peripheral immune cells, microglia can shift from M1 to M2 phenotype.

To inhibit the pro-inflammatory damage through M1 activation of microglia, its downstream signaling pathways could be targeted. The M1 phenotype is induced by IFN-γ via the JAK/STAT signaling pathway and targeting this pathway may arrest M1 activation. In fact, studies show that inhibition of the JAK/STAT pathway leads to suppression of the downstream M1-associated genes in several disease models. Another approach to suppress M1 activation would be to target the pro-inflammatory cytokines such as TNF-α, IL-1β and IFN-γ, and decrease its ability to interact with its receptors on other cell types. Alternatively, molecules with the capability to activate the anti-inflammatory M2 phenotype or promote the transition of pro-inflammatory M1 phenotype to anti-inflammatory M2 could be useful in the treatment of PD. Anti-inflammatory molecules such as IL-10 and beta interferons produce neuroprotection by altering the M1 and M2 balance.

The critical role of microglia in most neurodegenerative pathologies including PD is increasingly documented through many studies. Until recently, microglial activation in pathological conditions was considered to be detrimental to neuronal survival in the substantia nigra of PD brains. Recent findings highlight the crucial physiological and neuroprotective role of microglia and other glial cells in neuropathological conditions. Studies on anti-inflammatory treatments targeting neuroinflammation in PD and other diseases by delaying or blocking microglial activation failed in many trials due to the lack of a specific treatment approach, possibly the stage of disease and an incorrect understanding of mechanisms underlying microglial activation. With the updated knowledge on different microglial activation states, drugs that can shift microglia from a pro-inflammatory M1 state to anti-inflammatory M2 state could be beneficial for PD. The M1 and M2 microglial phenotypes probably need further characterization, particularly in PD pathological conditions for better therapeutic targeting. We support targeting of microglial cells by modulating their activation states as a novel therapeutic approach for PD.

This open access paper is an interesting companion piece to yesterday's discussion of the potential for expansion of mutations in stem cell populations to contribute to degenerative aging. What evolutionary constraints have led to the present state of stem cell populations in mammals: why are they not larger, with more capacity for tissue maintenance and regeneration in later life, for example?

Multicellular organisms continually accumulate mutations within their somatic tissues, constituting a significant, but poorly quantified, burden on tissue maintenance. To investigate this burden in a specific, well-parameterized context, we model the mammalian intestine and quantify the expected impact of mutation accumulation in stem cell populations. Furthermore, we explore how the population size of the stem cell niche influences mutation accumulation and demonstrate the expected trade-off between the risk of accumulating deleterious mutations, population size, and the risk of tumorigenesis. However, we further characterize how this trade-off can be expected to manifest over the lifetime of two well-studied mammalian systems, mice and humans, by estimating the expected effect of mutation accumulation on cellular homeostasis.

The intestinal epithelium is in constant flux, with populations of stem cells distributed throughout the intestine differentiating into other, transient, cell populations. These stem cells exist within small discrete populations in intestinal crypts, a compartmentalization thought to have evolved as a mechanism to deter tumorigenesis, as cells accumulating mutations that are beneficial to cellular fitness have a physical hindrance to spreading throughout the tissue. However, small populations are subject to significant genetic drift, that is, random changes in allele frequency that eventually lead to fixation or loss, and less effective selection.

The accumulation of damage causing the loss of cellular fitness is a hallmark of aging and is especially relevant when DNA damage occurs in stem cells, compromising their role in tissue renewal. Indeed, several mouse models with the diminished ability to maintain cellular genome integrity succumb to accelerated age-related phenotypes through the loss of tissue homeostasis caused by stem and progenitor cell attrition. Just as stem cell mutations conferring a beneficial fitness effect will increase cell production, mutations conferring a deleterious fitness effect will lead to decreased cell production and the diminished maintenance of healthy tissue.

When mutations confer a selective advantage or disadvantage within the niche, there exists an intermediate crypt size that minimizes the probability that any crypt accumulates the large beneficial mutations necessary to initiate a tumor. By modeling the fixation of mutations drawn from a full distribution of mutational effects and accumulating throughout the populations of the entire intestinal epithelium, we show that a secondary trade-off exists - populations maintained at a size that results in the lowest rate of tumorigenesis are expected to accumulate deleterious mutations that manifest in tissue attrition and contribute to organismal aging.

At small stem cell niche sizes, there exists a large number of crypts to maintain homeostasis, and a higher probability that any one crypt will obtain a rare mutation of large effect that would result in tumorigenesis. As stem cell niche size increases, the number of crypts needed to maintain the same amount of epithelium decreases, and so does the probability of fixing mutations within the crypts, and therefore the chance of fixing a rare mutation of large effect. However, for larger values of stem cell niche size, the strength of selection increases, thus increasing the chance that a fixed mutation was beneficial, leading to higher chances of tumorigenesis. At the observed intermediate population size in mice, the whole tissue size is expected to decline with age as deleterious mutations accumulate in stem cell niches. If selective pressures against tumorigenesis have selected for intermediate stem cell niche population sizes in mammalian species, then it has been at the expense of increasing epithelial attrition.

Link: https://doi.org/10.1111/eva.12476

Oisin Biotechnologies is a senescent cell clearance company founded by long-standing members of our community, seed funded by the Methuselah Foundation and SENS Research Foundation, and supported by the investment of a number of folk in the audience here. Targeted removal of senescent cells is a form of narrowly focused rejuvenation, shown to turn back numerous measures of aging in animal studies, and the Oisin team has made great strides in proving out their programmable gene therapy approach. This sort of commercialization project is exact what our community has been working towards all these years, and the faster that implementations reach the clinic, the better off we all are.

Oisin Biotechnologies' ground-breaking research and technology is demonstrating that the solution to mitigating the effects of age-related diseases is to address the damage created by the aging process itself. Our first target is senescent cells. When cells detect that they have been irretrievably damaged, they enter a non-dividing condition known as cell-cycle arrest, or senescence. It's believed this occurs to prevent cells from going rogue and turning cancerous. Ideally, they should die by the process known as apoptosis, but as we age, more and more frequently they don't. They become zombie cells - unable to kill themselves or resume normal function.

Senescent cells secrete molecules that cause inflammation in an effort to attract immune cells that would usually clear them. But for reasons that are not fully known, as we age, persistently senescent cells accumulate, leading to a vast number of age-related diseases. Oisin is developing a highly precise, patent-pending, DNA-targeted intervention to clear these cells. As a recent study has shown, clearing senescent cells both reduces negative effects of aging pathologies and also extends median lifespan and survival.

There are two major challenges to clearing senescent cells using our approach. First is to design and create the DNA construct that recognizes that a cell has become senescent, and then destroys it. Second is to safely and efficiently deliver this construct into cells throughout the body. Both goals have been achieved in our pioneering proof of concept experiments in 2016. We've first demonstrated the ability to transduce cells both in vitro (cell culture) and in vivo (in aged mice). Then we showed that p16 positive senescent cells can be killed on demand in both in vitro and in vivo environments. Now we are embarked on experiments that will show improvements in both healthspan and lifespan in model organisms from mice to primates. And then, everything changes.

Our proprietary technology gives persistently senescent cells a helping hand to "do the right thing." By providing an exogenous apoptotic gene, which is only transiently expressed in cells that already have the p16 gene active, we can precisely induce the senescent cell to commit suicide. Oisin has shown as much as an 80% reduction in senescent cells in cell culture and significant reductions of senescent cell burden in naturally aged mice. SENSOlytics is an Oisin proprietary platform technology that enables precise targeting of a senescent cell based on the DNA expression of the cell, not on surface markers or other characteristics that might be shared with normal, undamaged cells.

Link: https://www.oisinbio.com/

The immune cells known as macrophages are involved in debris cleanup and destruction of potentially harmful cells, among other tasks, but in recent years more attention has been drawn to the important role they play in the complex coordination of cellular activities relating to healing and tissue maintenance. It is even thought that a significant portion of the difference between limited human regeneration and proficient regeneration of the sort observed in salamanders might be explained by differences in macrophage behavior between these species.

Further, and possibly a near-future basis for therapies, macrophages involved in regenerative processes appear split into a few different classes with distinct behaviors and protein signatures. Although this is a case of arbitrary dividing lines drawn on a continuous spectrum rather than a case of clearly separate camps, it is a still a useful distinction to make. These types are known as polarizations, and the polarizations of interest in this discussion are M1 and M2. Both play important roles in the bigger picture, but M1 macrophages are generally less helpful in regeneration, spurring inflammation and fibrosis, while M2 macrophages are generally more helpful, suppressing inflammation and generating a supportive environment for regrowth.

Researchers are finding that it is possible to enhance the outcome of regeneration by increasing the ratio of M2 macrophages to M1 macrophages. More M2 macrophages and fewer M1 macrophages produces more rapid, more effective regrowth of tissues, and in some cases induces regrowth that normally doesn't occur with any reliability in mammals. This has been achieved in animal studies of nerve regeneration and bone healing, to pick a few examples. Interestingly, the cancer research community is interested in turning the dial in the opposite direction, generating more of the aggressive, inflammatory M1 macrophages that destroy cancer cells. As I said, both types have their part to play in the bigger picture.

Moving on to the topic at hand here today, the research below covers the impact of polarization on another part of the macrophage task list, that of debris clearance. Atherosclerosis is a condition in which oxidized, fatty metabolic waste enters the blood stream and sufficiently irritates a section of the blood vessel walls for the cells there to take action. Inflammatory signals draw macrophages that attempt to clean up the garbage, but macrophages are unfortunately unexpectedly frail in the face of this sort of fatty debris. Some ingest too much and either die or become senescent, dysfunctional foam cells, further aggravating the situation. Over time, a small irritated portion of a blood vessel wall swells into a self-perpetuating disaster zone of dead and dying macrophages. Eventually this happens somewhere critical, and driven by the hypertension of aging, a blood vessel wall or the fatty mass inside the vessel ruptures to cause a stroke or heart attack. It turns out that here, as elsewhere, it is the case that adjusting the natural balance towards more M2 and fewer M1 macrophages produces better outcomes, but just how useful this is for human medicine remains to be determined with any great certainty.

Mechanism Shown to Reverse Disease in Arteries

A certain immune reaction is the key, not to slowing atherosclerosis as cholesterol-lowering drugs do, but instead to reversing a disease that gradually blocks arteries to cause heart attacks and strokes. The study in mice focuses on reversing the effects of "bad cholesterol," which is deposited into the walls lining blood vessels in levels influenced by both genetics and a person's diet. By the fourth decade of life, and thanks to the chronic reaction to cholesterol, most people have inflamed "wounds" in their arteries, called plaques, which when severe enough can rupture to cause blood clots that block arteries. "Even the latest, most potent cholesterol-lowering drugs, PCSK9 inhibitors, let alone widely used statins, cannot fully reverse damage done to arteries over time. We need the next generation of drugs to go beyond cholesterol lowering to address the immune reaction to accumulated cholesterol, and to dismantle plaques as part of reversing or regressing mature disease."

Once deposited into arteries, bad cholesterol - known to physicians as low density lipoprotein - triggers the body's immune system, which is meant to destroy invading microbes but can drive inflammatory disease in the wrong context. Immune cells in the bloodstream called monocytes swarm to cholesterol deposits, and become either inflammatory or healing cell types based on signals there. In situations where disease is worsening in a plaque, past studies have shown that monocytes become M1 macrophages that amplify immune responses, increase inflammation, and secrete enzymes that gnaw at plaques until they rupture. The current study confirmed that monocytes arriving in plaques where disease is regressing instead become M2 "healing" macrophages, which dampen inflammation and prevent the ruptures that precede clotting.

When mice were engineered to lose the ability of monocytes to become M2 macrophages, they could no longer achieve normal disease regression. By surgically transplanting plaques from diseased mice into the arteries of healthy mice, the research team brought about dramatic drops in cholesterol levels. This drop has been shown to trigger a second benefit in mice, where monocytes automatically become M2 instead of M1 macrophages as plaques rapidly regress. It is not known whether cholesterol lowering alone triggers this M2 switch in humans, but new imaging techniques may soon be able to detect changes in the type and number of macrophages in plaques. In the meantime, if researchers learn how to boost the M2 switch, a number of clinical applications may become possible just as methods arrive that can measure their success. "A race is underway to develop treatments that enhance the decision of human monocytes to become M2 macrophages in cases where the disease has not yet caused clot formation, at which point it becomes irreversible."

Inflammatory Ly6Chi monocytes and their conversion to M2 macrophages drive atherosclerosis regression

Using a number of mouse models of atherosclerosis regression, including the aortic arch transplant used in the present study, we have previously shown that aggressive lipid lowering promotes the resolution of plaque inflammation, which is characterized by a decreased content of macrophages and an increase in the level of markers of the M2 state. We now extend these findings to show that plaque regression and the attendant resolution of inflammation surprisingly require the recruitment of new monocytes, which assume the characteristics of M2 macrophages. Furthermore, contrary to the prevailing paradigm, the newly recruited monocytes are drawn from the Ly6Chi circulating subset, generally considered to be "inflammation-prone" precursors of M1 macrophages.

The characteristic rapid reversal of hyperlipidemia in mouse atherosclerosis regression models is likely to reduce the continuous stimulation of the plaque inflammatory response by atherogenic lipoproteins, but clearly is not sufficient for the resolution of inflammation. Based on our results, M2 enrichment must also occur, and how the change in lipoprotein environment causes this to happen also remains to be determined. Our finding that it depends on STAT6-dependent signaling in the newly recruited monocytes suggests that local factors in the regressing plaque stimulate this signaling pathway. STAT6 is activated by two key cytokines, IL-4 and IL-13. However, which of these cytokines is the main player, as well as their cellular source(s), in promoting plaque regression is unclear.

Though many questions remain, the present results provide insights into the dynamic nature of the inflammatory process and the role of Ly6Chi monocytes in plaques. These cells were previously thought to contribute only to plaque progression and inflammation, but are now shown here to be important in regression and inflammation resolution. One clinically relevant insight raised by our studies is that strategies that promote the accumulation of M2 macrophages in atherosclerotic lesions may be a promising approach toward promoting plaque regression, consistent with recent studies in mice in which treatment with IL-13- or IL-4-based therapy was protective against atherosclerosis progression.

Mitochondria, the power plants of the cell, bear their own DNA, a small remnant of their origin as symbiotic bacteria. Unfortunately, this DNA is more vulnerable than the DNA found in the cell nucleus, and can become damaged in ways that contribute significantly to the aging process. How to address this problem? Allotopic expression of a mitochondrial gene is a process by which an altered version of the gene is placed into the cell nucleus in order to provide a backup source of the protein encoded by the gene. In this age of genetic engineering, inserting the gene isn't really the challenge, instead the difficulty lies in figuring out how to alter the gene in order for the protein produced to be transported back to the mitochondria where it is needed.

Funded by philanthropic donations, the SENS Research Foundation has been supporting allotopic expression research for a decade now, seeking to accelerate the development of therapies that can remove this contribution to the aging progress. The first programs funded gave rise to Gensight Biologics, a company that is pioneering the use of allotopic expression of the ND4 gene to address an inherited blindness condition in which the gene is mutated and dysfunctional. This effort is well on the way to proving out the technology in human trials and thereby providing a solid foundation for work on the other genes that must be backed up. Three mitochondrial genes are demonstrated so far, including ND4, and there are another ten to go after that. Commercial efforts of this nature are an important part of the overall development process, and it is a good thing to see a company pulling in significant funding for a technology that will become a part of later rejuvenation therapies.

GenSight Biologics has raised €22.5 million to prepare to bring gene therapy GS010 to market in the U.S. and Europe. The financing gives the Novartis-backed biotech enough cash to deliver data from two phase 3 trials next year and gear up for anticipated approvals on both sides of the Atlantic. Paris-based GenSight raised the cash from a mix of new and existing institutional investors, most of which are based in the US. Strong interest from these backers saw GenSight ease past its initial target of €20 million to pull in €22.5 million in the private placement. When added to the €48.8 million GenSight had in the bank at the end of March, management thinks the money moves its runway out to the first quarter of 2019.

That runway covers a critical period for GenSight. Topline 48-week data from two phase 3 trials of GS010 in patients with Leber hereditary optic neuropathy (LHON) are due in the second and third quarters of next year. GenSight is looking to the trials for evidence GS010 improves the clarity of the vision of patients with LHON, a hereditary form of vision loss caused by mitochondrial defects. GS010 is injected into the eye to deliver the human wild-type ND4 gene via an adeno-associated virus to deliver. This gene encodes for a protein typically produced by mitochondria.

One trial is assessing GS010 in patients who started losing their vision in the six months prior to enrolling in the study. The other is recruiting patients whose vision started deteriorating between seven and 12 months ago. Both trials are injecting GS010 into one eye of each participant and pretending to inject it into the other eye. Data from an earlier phase 1/2 trial suggest the gene therapy is most effective in patients whose vision started deteriorating less than two years ago. A recent 96-week update found the treated eyes of such patients had a mean gain of 29 ETDRS letters, as compared to an increase of 15 letters in untreated eyes. ETDRS is the test showing progressively smaller letters opticians use to gauge vision. The performance of GS010 to date has enabled GenSight to secure the support of some big-name backers. Following the latest financing, its biggest shareholders are Novartis, Versant, Abingworth, and Fidelity.

Link: http://www.fiercebiotech.com/biotech/novartis-backed-gensight-gets-cash-for-gene-therapy-launch

To what degree does random mutation in nuclear DNA contribute to aging over the present human life span? The present consensus is that this is a cause of disarray in metabolic processes, and that it does reach a significant level of consequence for tissue function. Unfortunately there is little direct evidence for this view - it is hard to split out just nuclear DNA damage from the rest of aging in order to isolate its effects, though there a few lines of research showing promise in this direction. Researchers here take a different approach to the question; they suggest that some forms of random mutational damage that occurs in stem cells will expand throughout that population over time, because the damage in some way confers a replication advantage. In this way it can come to have a significant effect, and it should be feasible to correlate different degrees of the expansion of this sort of mutational damage with specific measures of aging. In this case, the correlation is with cardiovascular disease.

Several explanations have been offered for how age contributes to cardiovascular disease. Aging is associated with the acquisition and exposure duration of other established risk factors for cardiovascular disease, including high systolic blood pressure and increased levels of low-density lipoprotein cholesterol. However, analyses that adjust for the concomitant burden of other risk factors consistently identify age as an independent predictor of cardiovascular disease. Modifiable risk factors account for only about 12% of the age effect in men and 40% in women. Thus, the aging process itself must promote cardiovascular risk, although the mechanisms that are involved are poorly understood.

Researchers now provide new insight into how aging can promote atherosclerosis and cardiovascular events in their investigation of a phenomenon termed clonal hematopoiesis of indeterminate potential, or CHIP. This condition is an age-related disorder characterized by the acquisition of somatic mutations in hematopoietic stem cells that confer on these cells a selective advantage. As a consequence, instead of the normal polyclonal generation of blood cells, mutation-containing clones expand over time and make up an increasing percentage of the stem cells and their progeny and may include granulocytes, lymphocytes, and monocytes. CHIP is rarely found in patients who are younger than 40 years of age, whereas this condition may exist in up to 10% of persons over the age of 70 years. Patients with CHIP have a higher rate of death from noncancer causes (particularly cardiovascular disease) than do age-matched controls without CHIP.

To address the cause of excess cardiovascular mortality, researchers identified CHIP (which they define as clonal dominance of hematopoietic cells bearing pathogenic mutations in any of 74 known driver genes of hematologic cancers) among participants in several studies that ascertained cardiovascular disease. In studies involving participants with a mean age of 60 years or older, carriers of CHIP had nearly twice the risk of coronary heart disease as noncarriers. Among younger participants (below 50 years of age), CHIP carriers had four times the risk of myocardial infarction as noncarriers. Preclinical coronary disease, as assessed on imaging as coronary-artery calcification, was also associated with CHIP. Finally, four of the most commonly mutated genes in CHIP (DNMT3A, TET2, ASXL1, and JAK2) were each individually associated with coronary heart disease.

Collectively, the work supports the hypothesis that CHIP is linked to the clinical events of atherosclerosis and that certain CHIP driver genes are involved in regulating inflammation. Both TET2 and DNMT3A appear to inhibit inflammation, so loss-of-function mutations in these genes could plausibly promote inflammatory responses. Similarly, there is a large body of literature suggesting that JAK2 regulates both inflammation and thrombosis, two important factors in the clinical manifestations of atherosclerosis. Thus, the data is consistent with established paradigms that inflammation is an accelerator of atherosclerosis and coronary heart disease. Moreover, their findings should prompt a discourse about studying the use of anti-inflammatory agents in patients with CHIP to limit the most common cause of death in these patients - cardiovascular disease.

Link: https://doi.org/10.1056/NEJMe1706173

Today I'll point out an interesting paper on the demographics of aging, one that I hope indicates the spread of more nuanced and useful views into forecasts of the future of aging and longevity. While a good read, and helpful for our cause in that it will further spread the message that increases in healthy life span are both realistic and currently taking place, it is nonetheless still the case that this and all of the other long-term projections arising from the demographic community are essentially fantasies. They are simple extrapolations of trends in adult life expectancy established over the past few decades, and are thus based on a model of the future in which methods of rejuvenation are never invented and commercialized. In this future, progress in medicine consists only of incremental improvements to the present marginally effective therapies that attempt to patch over or compensate for the damage of aging. These treatments fail to repair or otherwise address that damage in any meaningful way, which is precisely why they produce only marginal outcomes at best.

This proposed continuation of the present gentle upward trend in life expectancy will not come to pass. The trend was established across decades in which no therapy attempted to address the cases of aging, the accumulation of molecular damage to cell and tissue structures that produces age-related disease and degeneration. Consider that for any failing machinery, it is very hard to keep it running when unable to fix the root cause of that failure. This is also the case for our biology: damage accrues, causing progressively greater system failures that we experience as age-related disease. Aging is no longer inevitable, however, as the first generation of rejuvenation therapies are even now nearing the clinic. To pick one example, selective destruction of senescent cells has been carried forward by venture funded companies these past couple of years, with human trials starting soon. Further, successful trials have been carried out in the past few years for clearance of forms of amyloid from old individuals, and again there is sizable backing for those lines of development.

Old age in the future of deliberate efforts to repair the damage that gives rise to aging will be profoundly different from old age in the past, in which medicine failed to address the causes of aging. There will be an abrupt upward discontinuity in the past trend of life expectancy, a sudden and considerably faster rate of increase in years of health and vigor. It is the difference between doing nothing for the causes of aging versus doing something to tackle those causes, and here and now is the dividing line between those two periods of development in medicine.

New Measures of Aging May Show 70 is the New 60

Traditional population projections categorize "old age" as a simple cutoff at age 65. But as life expectancies have increased, so too have the years that people remain healthy, active, and productive. In the last decade, researchers have published a large body of research showing that the very boundary of "old age" should shift with changes in life expectancy, and have introduced new measures of aging that are based on population characteristics, giving a more comprehensive view of population aging. The study combines these new measures with UN probabilistic population projections to produce a new set of age structure projections for four countries: China, Germany, Iran, and the USA.

One of the measures used in the paper looks at life expectancy as well as years lived to adjust the definition of old age. Probabilistic projections produce a range of thousands of potential scenarios, so that they can show a range of possibilities of aging outcomes. For China, Germany, and the USA, the study showed that population aging would peak and begin declining by 2040 in Germany and by 2070 in China, well before the end of the century. Iran, which had an extremely rapid fall in fertility rate in the last 20 years, has an unstable age distribution and the results for the country were highly uncertain. Population aging - when the median age rises in a country because of increasing life expectancy and lower fertility rates - is a concern for countries because of the perception that population aging leads to declining numbers of working age people and additional social burdens.

Probabilistic population aging

Probabilistic population forecasts were motivated by Keyfitz. Keyfitz wrote: "Demographers can no more be held responsible for the inaccuracy in forecasting population 20 years ahead than geologists, meteorologists, or economists when they fail to announce earthquakes, cold winters, or depressions 20 years ahead. What we can be held responsible for is warning one another and our public what the error of our estimates is likely to be."

There is now an extensive literature of probabilistic forecasting. All the probabilistic measures of aging produced by the United Nations assume that the threshold of old age is a fixed chronological age, regardless of time, place, education, or other characteristics of people. Researchers have questioned this assumption: "To the extent that our concern with age is what it signifies about the degree of deterioration and dependence, it would seem sensible to consider the measurement of age not in terms of years elapsed since birth but rather in terms of the number of years remaining until death." It is suggested the old age threshold be defined on the basis of some plausible remaining life expectancy rather than any specific chronological age. We call the ages, that are obtained when life expectancy is the characteristic that is held constant, prospective ages and measures that use them prospective measures of population aging.

New measures of population aging are useful because tomorrow's older people will not be like today's. They may well have longer life expectancies, better cognition, better education, and fewer severe disabilities. In most OECD countries, the labor force participation of people 65+ years old is increasing as are the ages at which people can receive a normal national pension. Since changes in the characteristics of people are ignored in the conventional measures of aging, they become more outdated with the passage of time. The use of prospective ages is one way to create measures of aging that are more in line with observable changes.

We chose a remaining life expectancy of 15 years. That was the life expectancy at age 65 in many low mortality countries around 1970. We show estimated and forecasted old age thresholds based on a remaining life expectancy of 15 years for China, Germany, Iran and the US for the years 2013 through 2098. In 2013, the old age threshold was 66 in China and 72 in Germany. By 2098, the old age threshold is forecasted to increase to 79 in Germany and 77 in China. Most of the population aging that we measure in China, Germany, and the US occurs between now and around 2040. The probabilistic forecasts show that it is highly likely that the prospective median age of the population of Germany and the US will be lower in 2098 than it is today. Even in China there is around a 50 percent chance that the prospective median age will be lower at the end of the century than it is today. It is possible that people's concern about the future is related to the number of additional years they expect to live. Lower prospective median ages in 2098 than now indicate that the people at the median age will have even more years of additional life ahead of them than people at the median age have currently, despite that median age being higher.

The development of a reliable and accurate biomarker of biological age is an important step for the longevity science field. Testing potential rejuvenation therapies is at present a drawn-out and expensive process, as the only truly effective way to determine outcomes is to wait and see. That requires years and millions of dollars in funding for mouse studies, a cost that greatly restricts the amount of experimentation and exploration it is possible to carry out, even for the better funded research groups. If instead a biomarker test could be applied shortly before and shortly after a treatment in order to assess its potential, that would greatly accelerate progress in the field. Epigenetic clocks based on assessment of patterns of DNA methylation are presently the most promising candidate for such a biomarker of aging, and here researchers discuss the behavior of presently established clocks in mice and humans.

Epigenetic clocks provide powerful tools to evaluate nutritional, hormonal, and genetic effects on aging. What can we learn from differences between species in how these clocks tick? One of the most fascinating findings in human aging is that it is associated with highly reproducible DNA methylation (DNAm) changes. DNAm levels at age-associated CG dinucleotides (CpG sites) can be integrated into epigenetic age predictors, which provide robust biomarkers to estimate chronological age. With the advent of more and more publically available DNAm profiles, such aging signatures were further developed to facilitate higher precision in age predictions, particularly for blood samples. Probably the most commonly used epigenetic aging signature is based on DNAm levels at 353 CpG sites and facilitates relatively precise age predictions for many human tissues: the median "error" (MAE), defined by the median absolute difference between DNAm age and chronological age, is usually less than 4 years.

Now - about 6 years after the first epigenetic clock paper - similar age predictors have been established for mice. Again, they were initially described for defined murine tissues, specifically liver and blood, taking into account the fact that there are notoriously large differences in the epigenetic makeup of cells from different tissues. However, it is also possible to derive a multi-tissue murine DNAm age predictor, in analogy to the most commonly used human clock. The signature is based on 329 CpGs and has been validated for cortex, muscle, lung, liver, and heart tissue. Overall, the multi-tissue age predictor reached a MAE of less than 4 weeks, although how it performs in other tissues has yet to be shown.

Studies indicate that the epigenetic clocks of mice tick faster than those of humans. This can be anticipated because the maximum life-span of mice (about 2 years) is much shorter than it is in humans (about 85 years). If the molecular changes of aging are linked to life expectancy and generation time, then this might support the notion that aging reflects a controlled evolutionary process. However, there is still an open debate on whether aging is due to an accumulation of cellular defects, or is driven by a developmental mechanism. Either way, comparison of epigenetic clocks in mice and men will provide new insights into the regulation of age-associated DNAm.

Direct comparison of age-associated CpGs in mice and men indicated that there is a moderate but significant association between the two species. It is not always trivial to identify orthologous CpG sites, and further interspecies comparison will be required to better understand similarities and differences of age-associated genomic regions. However, the overlap of age-associated CpGs in age predictors for human and mice seems to be rather low, and hence epigenetic clocks need to be trained specifically for different species. In terms of function, age-associated CpGs in humans and mice seem to be enriched in genes that are involved in morphogenesis and development. However, in both species age-associated DNAm changes are not generally reflected at the gene expression level - and thus the biological relevance remains largely unclear.

Mouse DNAm clocks provide powerful tools to study longevity interventions in one of the most relevant model organisms for aging research. These signatures were initially trained to correlate with the "real" chronological age of mice - but aging rates may differ between individuals. In fact, there is evidence that epigenetic clocks rather reflect the biological age, which is related to the perceived aging process of an organism. In analogy, it was previously demonstrated that human DNAm age is related to life expectancy: accelerated epigenetic age is associated with higher all-cause mortality. This finding has been validated in various additional cohorts and with different epigenetic age predictors. Furthermore, human epigenetic aging rates have been shown to be significantly associated with sex, race/ethnicity, and some disease risk factors. In mice, there was no clear difference in predicted DNAm age of male and females. However, ovariectomy, which reduces the average life span in female rats, results also in significant age acceleration. Caloric restriction or dietary rapamycin treatment, both of which result in increased life expectancy of mice, reduced epigenetic age. In humans, specific diet seems to have a less pronounced impact on epigenetic age, but there is significant association of DNAm age and body mass index (BMI). Apparently, different parameters can affect biological aging in mice and men.

Link: https://doi.org/10.1186/s13059-017-1245-8

Following on from a recent post on the subject, here is another article from the Life Extension Advocacy Foundation (LEAF) on strategies and efforts to persuade the world to support work aimed at greatly lengthening healthy human life spans. For those of us to whom it is obvious that a very large amount of time and funding should be devoted to this goal, because aging is by far the greatest source of suffering and death, because the cost of bringing aging under medical control is small in comparison to what is spent on trying and failing to cope with it, and for a score of other equally good reasons, it can be frustrating to see that others do not presently think that way. They seem happy to march to their deaths, while at the same time happy to avail themselves of the small gains resulting from the comparatively crude medicine for age-related disease that is presently available. If rejuvenation therapies existed, these same people would use them and be thankful for them, but very few will so much as raise a finger to help create these technologies. It is a challenge.

One of the most frequent questions we at LEAF hear is this: when will innovative therapies to delay, stop and eventually reverse age-related damage become available? This is not an easy question to answer, because the pace of progress depends on many factors - predominantly, funding. Fundamental studies on aging are not well-funded and the accumulation of knowledge necessary to proceed from lab work to clinical trials and then clinical practice does not happen fast enough. Government funding is more often allocated to mainstream areas, such as research on single diseases. Business, for its part, does not show much interest in fundamental science, because usually there is no final product that can be sold. The only other source of funding is the general public. But most people are not yet sufficiently informed about the plausibility of bringing the aging processes under medical control and do not share the values of our community. Sometimes (to be frank, pretty often) activists trying to engage the public in an enlightening conversation can encounter resistance or even rejection.

Learning requires active, conscious participation. This means that students will seek and absorb information if they are interested, or they will ignore it if they do not see any personal benefit in it. So what do most people want? It's life extension right? Wrong! Studies show that when people are asked "how long would you like to live?" with no other conditions specified, people added around 5 to 10 years to the average life expectancy for their country and that was it. If we do not first explain the aging processes, the connection with age-related diseases and the repair based solutions that lead to healthy longevity, people think longevity means a longer life of prolonged ill health and frailty. Who wants to spend another ten or twenty years in a wheelchair or in a care home? And this is exactly the image most people have when these words are used. This is what the expression "life extension" means to the majority of people - and this is why we should avoid beginning the conversation with it.

At the same time, sociological studies show that if the possibility of perfect health throughout life is introduced into the equation from the very beginning, people show much more interest and support for the idea of ​​prolonging life. People literally switch from one camp to the other, those who just did not want to live for more than 80 years now decide they want to live to more than a hundred, and those who just wanted to get to 120 are suddenly ready to live to 150 or beyond. People are ready to support the development of new medical technologies, even scary ones like gene therapies or gene editing, under the condition they are going to be used for treatment. However their use for the prolongation of life belongs to the category of "human enhancement" and as such the idea is most often rejected. So it is highly recommended for advocates to add that these technologies will help treat or prevent serious chronic diseases, while extended lifespans will just be a possible nice side-effect.

Link: http://www.leafscience.org/effective-advocacy/

The SENS Research Foundation annual reports tend to arrive in the middle of the following year, and today the 2016 report was published. You can find it in PDF format at the foundation website. The story of SENS rejuvenation research, approaches that aim to repair the cell and tissue damage that causes aging, is one of growth and success over the years. It has been a bootstrapping from idea to reality, powered by the philanthropy and determined support of our community. We have come a long way and achieved a great deal these past fifteen years. Yet there remains the upward curve ahead, and the completion of the vision of an end to aging has yet to be accomplished.

It is true that the first SENS therapies are on the way to the clinic, their commercial development funded by venture capital now, and senescent cell clearance is at the front of the pack. But equally important approaches to removing the damage that causes aging, such as the breaking of glucosepane cross-links, are still in the laboratory, still entirely funded by charitable donations, still building the infrastructure and running the tests in search of the first potential basis for a working therapy. When that first breakthrough is made, matters speed up considerable and funding comes running from many sources - but getting there is a slow grind. The more we can do to help the SENS Research Foundation thrive, the faster they can push forward with this stage of development: planting the seeds that will blossom into vast medical industries, and in doing so bring great benefit to humanity.

SENS Research Foundation 2016 Annual Report (PDF)

Since SENS Research Foundation's founding in 2009, we've worked toward bringing our vision of a world free of age-related disease from concept to reality. In challenging ourselves on this front, we have likewise challenged you, our supporters. We've asked a lot of all of you, and not only have you accepted this challenge, you have delivered. The rejuvenation biotechnology community that has emerged over the past several years owes its existence to each and every one of you. You have become our most vocal advocates. Over 2000 of you have become our funders.

We asked you to help us change how the world researches and treats age- related disease. You did. Through the efforts of our donors, collaborators, and our advisory board, world-renowned institutions are pursuing age- related disease research specifically focused on the damage-repair paradigm. We asked you to help us move from basic research to translational research and clinical trials. You did. In 2016 we launched Project|21, our five-year plan to help move rejuvenation biotechnologies from concept to human clinical trials. Project|21 is now backed by a number of generous and forward-thinking individuals.

You asked us to follow through. We did. In lending your support, you place not only resources in our hands, but trust. We know that a world-changing nonprofit cannot operate on the power of vision alone; and we are here not just to inspire, but to deliver results. The purpose of this report is to demonstrate concrete examples of those results to you. With your help, we have taken great steps toward the establishment of a robust rejuvenation biotechnology industry and the realization of our vision. And every step we are able to take is proof of the power of your community.

Death-Resistant Cells: Toward Neutralizing the SASP

Buck Institute researchers led by Dr. Judith Campisi had shown that the presence of senescent cells alongside cancer cells can stimulate those cells to both multiply more rapidly and to spread to other parts of the body - the metastasis process, which ultimately makes most cancers so deadly. Repeating these studies in cell culture while inhibiting the senescence-associated secretory phenotype (SASP) with apigenin almost completely nullified the proliferation-stimulating and pro-metastatic effects of senescent cells on breast cancer cells. Drugs based on parts of apigenin's structure could dampen some of the harmful effects the SASP in senescent cells. Removing these cells is the ultimate solution to these problems, and in the last year several groups have made rapid progress toward this goal. In the meantime, these studies using apigenin may demonstrate important principles from which senescent-cell-focused rejuvenation biotechnologies may be derived.

Target Prioritization of Tissue Crosslinking

Our arteries slowly stiffen with age, in substantial part because of random crosslinking of the structural proteins collagen and elastin. Developing rejuvenation biotechnologies to break these crosslinks is key to restoring youthful arterial function. To tease out the effects and relative importance of all of these different sources of crosslinking in aging tissues, the Babraham Institute team has been studying the crosslinking process in the tissues of aging mice. This has required the development and validation of new experimental methods and assays, which are now ready for use. The team has evaluated multiple tissues for crosslink presence. Importantly, some of the crosslinks that have been reported by others to accumulate in aging tissues were not detected. While further studies are needed to confirm it decisively, these results suggest that several crosslinks now believed to accumulate in aging tissues may actually be experimental artifacts.

Engineering New Mitochondrial Genes to Restore Mitochondrial Function

Free radicals derived from our energy-producing mitochondria can mutate the organelle's DNA, leading to deletions of large stretches of the mitochondrial genome. These deletion mutations prevent the mitochondria from building various pieces of the electron transport chain (ETC), with which mitochondria generate most cellular energy. The accumulation of deletion-mutation-containing cells is a significant consequence of aging. A potential rejuvenation biotechnology to recover ETC function is the allotopic expression of functional mitochondrial genes: placing "backup copies" of all of the protein-coding genes of the mitochondria in the "safe harbor" of the nucleus, thereby giving the mitochondrion all of the proteins it needs to continue producing energy normally even when the original mitochondrial copies have been mutated.

This year, the SRF MitoSENS team reported a tremendous success: for what they believe is the first time, they have used allotopic expression to rescue the complete loss of a mitochondrially-encoded protein in a mammalian cell. A publication announced their success in the fall of 2016. The results show that their targeted and recoded ATP8 protein can be expressed from the nucleus, turned into protein in both normal and mutant cells, and efficiently targeted to the mitochondria. Furthermore, they can demonstrate functional rescue of cells. Under conditions where mutant cells die for lack of ability to produce energy, the cells with engineered allotopically-expressed proteins were able to survive and replicate. In addition to ATP8, the SRF MitoSENS team has further demonstrated expression and targeting of a second re-engineered protein, ATP6. It is proof-of-concept that ATP8 is not a special case.

Identification of the Genetic Basis of ALT in Cancer

Telomeres shorten every time a cell divides, and thus all cancers have to find a way to keep their telomeres long enough to prevent senescence or death. Most cancers use an enzyme called telomerase for this purpose, but about 10-15% of cancers use a telomerase-independent mechanism known as Alternative Lengthening of Telomeres (ALT). The ALT mechanism remains largely a mystery, and therefore the OncoSENS team at SENS Research Foundation is working hard to find new ways to attack ALT cancers. First, the team has developed and established two separate high-throughput assays measuring different ALT-specific biomarkers. These assays will finally enable cancer researchers to screen hundreds of thousands of compounds across multiple drug libraries, or even test every single one of the more than 20,000 genes in the human genome, for ways to shut down ALT cancers. In addition to their biomarker work, the team is also pursuing more targeted methods to kill ALT cancer cells.

Glucosepane Crosslinks and Routes to Cleavage

One major cause of crosslink accumulation in aging is Advanced Glycation Endproducts (AGE), and one AGE in particular, called glucosepane, is currently thought to be the single largest contributor to tissue AGE crosslinking. The Yale AGE team is studying the role of AGEs in aging, and developing novel tools and strategies for reversing AGE-mediated protein damage and develop new antibodies and reagents to enable rejuvenation research. Our pilot lab at Cambridge University found that all of the commercially-available antibodies for the major AGE-related molecules are actually highly unreliable. This is a serious impediment. The Yale glucosepane team is now tackling this problem via the novel chemistry and methods they have developed. In the last twelve months, the Yale team has made exciting progress in their work. Most notably, they have developed the first synthetic route to produce glucosepane. Their novel synthetic strategy is the first ever to provide high yields of pure samples of glucosepane, putting them (and soon other scientists) for the first time in a position to explore mechanisms through which crosslinks can be broken.

In collaboration with a colleague at Yale, they have also developed a high-throughput assay for screening proprietary libraries of organic catalysts for agents capable of breaking synthetic glucosepane. One of these libraries has already been taken forward for proof of concept, which led to the identification of several leads for catalysts that could be capable of breaking glucosepane. Beyond that, the Yale group has successfully generated proteins containing their synthetic glucosepane that can be used to identify antibodies that label glucosepane-containing proteins. These antibodies will enable the immunochemical detection of glucosepane crosslinks for a wide range of applications.

Tissue-Engineered Thymus

The thymus gland is responsible for the development of a class of immune cells called T-cells. As part of the degenerative aging process, the thymus shrinks in size. This process of thymic atrophy prevents the body from maturing new T-cells, progressively weakening the immune system's ability to fight off never-before-encountered infections. Engineering new, healthy thymic tissue would help to restore the vigorous immune response of youth. SENS Research Foundation has therefore funded a Wake Forest Institute for Regenerative Medicine (WFIRM) group to apply tissue engineering techniques to the creation of functional thymic tissue to fortify or replace the aging thymus. Engineering new tissues requires a "scaffold" in which to embed cells to give them structure and functional cues, and the WFIRM group has tested different scaffolding systems: decellularized donor scaffolds and hydrogels.

In the decellularized scaffold paradigm, an organ of the type that is needed is taken from a donor, but is then stripped of its original cells and DNA, leaving behind a protein structure with low potential for immunological rejection that can be repopulated with cells taken from the new organ recipient. The WFIRM group initially began work in this paradigm using mouse organs, but they found that once decellularized, mouse thymuses lacked the rigidity to serve in that role. They accordingly moved on to the pig thymus - a species that not only worked well as an experimental system, but has some clinical potential as well. The pig is closer to humans both immunologically and in terms of size.

Catalytic Antibodies Targeting Transthyretin Amyloid

As part of the degenerative aging process, proteins that normally remain dissolved in bodily fluids become damaged, and adopt a misfolded form called amyloid. Amyloid composed of the transporter protein transthyretin (TTR) deposits in the heart and other organs with age, beginning to impair heart function. With SRF funding, the University of Texas-Houston Medical School (UTHMS) extracellular aggregate team is working to develop novel catalytic antibodies ("catabodies") that would recognize and cleave TTR amyloid deposited in the heart and other tissues. Catabodies have the potential to be safer and more effective than conventional antibody-based immunotherapies: their catalytic activity minimizes the amount of antibody required to clear deposits from tissues, and the fact that they don't form stable complexes with their targets or engage immune cells is expected to minimize the inflammatory side-effects seen with other experimental antibody therapies.

Work has resulted in the identification of two powerful TTR-cleaving catabodies. When tested for their ability to degrade misfolded wild-type TTR, these candidates were able to hydrolyze both soluble aggregates and deposit-like particulates, while having no effect on either TTR in its healthy, normal conformation or on a selection of fourteen other physiologically important proteins. Concentrations required to disintegrate 80% of a sample of amyloid were many hundreds of times lower than those routinely achieved in the blood using other infused antibodies. The establishment of stable cell lines will enable larger-scale production, as the team works to develop these candidates into functional rejuvenation biotechnologies.

Rejuvenation of the Systemic Environment

There might be a misunderstanding of what was really going on in parabiosis. When animals are connected, they are not just given reciprocal blood transfusions, but are surgically joined together. So in addition to receiving young blood, the old animals also have their old blood filtered through the young animals' livers and kidneys, and diluted with the young pairmate's own blood. Might the effects of parabiosis mostly come from the removal of toxic or suppressive factors from the old animals' sluggish circulation instead of from the delivery of active rejuvenating factors?

To test this possibility - and to accelerate identification and testing of potential pro- and anti-rejuvenation factors in the exchanged blood - SENS Research Foundation funded Dr. Conboy and the UC Berkeley systemic environment team's development of a novel technological platform. Using a mixture of off-the-shelf and custom 3-D printed parts, this platform enables the group to easily and safely extract blood from small animals and transfuse it quickly and directly into another animal, without the reciprocal exchange of its blood or the passage of its blood through the pairmate's system. It thus separates the effects of the young animals' metabolic and excretory systems from the pure effects of their blood.

The team then used the new system to repeat key parabiosis experiments from Dr. Conboy's and others' labs. As compared with the impact of full-on parabiosis, the effects of isolated young blood on old muscles' ability to repair an injury were still substantial: the stem cells recovered significant regenerative powers, and less residual fibrosis remained after the wound was resolved. But by contrast, previously-reported benefits of parabiosis in the brain and the liver were either not present, or were far more modest. Another critical finding was the confirmation of suppressive factors in the old animals' blood, which inhibited neurogenesis and other regenerative responses of young animals transfused with it. While this clearer picture of the basis of the "parabiosis effect" indicates a lower likelihood of isolating true pro-rejuvenation factors in the blood of young mice, we are nonetheless closer to being able to filter out factors responsible for suppressing the regenerative potential of an older body.

Commercial Development

Two of the companies SENS Research Foundation has supported are moving to raise funding to move their research from the lab to clinical trials. Ichor Therapeutics announced a Series A offering to bring its Lysoclear product for age-related macular degeneration and Stargardt's macular degeneration through Phase I clinical trials. In 2014, Ichor Therapeutics completed a material and technology transfer agreement for rights to concepts and research pioneered by SENS Research Foundation. Lysoclear, which Ichor announced in 2017, is a recombinant enzyme product based on extending SRF's prior work that selectively localizes to the lysosomes of retinal pigment epithelium cells where A2E accumulates, and destroys it. Ongoing studies suggest that Lysoclear is safe and effective at targeting A2E, the main toxin driving these diseases, eliminating up to 10% with each dose. This product would be the first clinical candidate based on concepts and research pioneered by SENS Research Foundation.

Oisin Biotechnologies is focused on the genetic elimination of unwanted cells, but without involving the immune system. Oisin reports significant progress in showing that their vector works, efficiently transducing cells and delivering a DNA construct which can kill targeted cells on command. Oisin closed a $500K oversubscribed convertible debt round in mid-December and is working towards a substantial Series A in the next few months that would take it towards a Phase 1 clinical trial.

Researchers here provide evidence for Parkinson's disease to have a significant autoimmune component, adding to other factors known to contribute to the death of dopamine generating neurons that is characteristic of this disease. It is certainly the case that the growing dysfunction of the immune system in later life includes a variety of autoimmune aspects, and that those aspects are still poorly mapped. It is reasonable to expect that researchers will continue to uncover ways in which immune system failures contribute to well-known age-related conditions in the years ahead. The disovery here is particularly interesting, as it links autoimmunity to the buildup of metabolic waste and consequent failure of mechanisms of maintenance that is observed in aging. This is a target for the SENS rejuvenation research program, and therapies built on this approach should prove broadly beneficial, precisely because they will halt and turn back many chains of consequences akin to that reported by the researchers here.

Researchers have found the first direct evidence that autoimmunity - in which the immune system attacks the body's own tissues - plays a role in Parkinson's disease, the neurodegenerative movement disorder. The findings raise the possibility that the death of neurons in Parkinson's could be prevented by therapies that dampen the immune response. "The idea that a malfunctioning immune system contributes to Parkinson's dates back almost 100 years. But until now, no one has been able to connect the dots. Our findings show that two fragments of alpha-synuclein, a protein that accumulates in the brain cells of people with Parkinson's, can activate the T cells involved in autoimmune attacks. It remains to be seen whether the immune response to alpha-synuclein is an initial cause of Parkinson's or if it contributes to neuronal death and worsening symptoms after the onset of the disease. These findings, however, could provide a much-needed diagnostic test for Parkinson's disease and could help us to identify individuals at risk or in the early stages of the disease."

Scientists once thought that neurons were protected from autoimmune attacks. However, in a 2014 study, researchers demonstrated that dopamine neurons (those affected by Parkinson's disease) are vulnerable because they have proteins on the cell surface that help the immune system recognize foreign substances. As a result, they concluded, T cells had the potential to mistake neurons damaged by Parkinson's disease for foreign invaders. The new study found that T cells can be tricked into thinking dopamine neurons are foreign by the buildup of damaged alpha-synuclein proteins, a key feature of Parkinson's disease. "In most cases of Parkinson's, dopamine neurons become filled with structures called Lewy bodies, which are primarily composed of a misfolded form of alpha-synuclein."

In the study, the researchers exposed blood samples from 67 Parkinson's disease patients and 36 age-matched healthy controls to fragments of alpha-synuclein and other proteins found in neurons. They analyzed the samples to determine which, if any, of the protein fragments triggered an immune response. Little immune cell activity was seen in blood samples from the controls. In contrast, T cells in patients' blood samples, which had been apparently primed to recognize alpha-synuclein from past exposure, showed a strong response to the protein fragments. In particular, the immune response was associated with a common form of a gene found in the immune system, which may explain why many people with Parkinson's disease carry this gene variant.

Researchers hypothesizes that autoimmunity in Parkinson's disease arises when neurons are no longer able to get rid of abnormal alpha-synuclein. "Young, healthy cells break down and recycle old or damaged proteins, but that recycling process declines with age and with certain diseases, including Parkinson's. If abnormal alpha-synuclein begins to accumulate, and the immune system hasn't seen it before, the protein could be mistaken as a pathogen that needs to be attacked."

Link: http://newsroom.cumc.columbia.edu/blog/2017/06/21/parkinsons-is-partly-an-autoimmune-disease-study-finds/

A good deal of evidence has accumulated to show that the immune cells called macrophages play important roles in regeneration. Further, there are several different classes of macrophage with quite different behaviors, and while all are essential in the bigger picture, one of them tends to hinder regeneration as a side-effect of the accomplishment of its other duties. Researchers have shown in a number of studies that adjusting the proportion of macrophages in a tissue, towards less of the hindering type, can significantly improve outcomes, and perhaps even produce regeneration that would normally not occur with any great reliability, such as regrowth of nerve tissue. This paper is a recent example of this area of research:

After nerve trauma, the standard clinical operating procedure is to oppose the two nerve ends and, when possible, suture them together. Ultimately, even with successful autografting, only 40% of patients regain useful function. Therefore, there is a clear, urgent, and unmet clinical need for an alternative approach that can match or exceed autograft performance. After peripheral nervous system (PNS) injuries, neurons respond rapidly by changing their activities and promoting a regenerative phenotype. At the distal nerve stump, Schwann cells (SCs) adopt a reparative phenotype. SCs, as well as infiltrating and resident macrophages, remove inhibitory debris, enabling new axons to sprout into the degenerated nerve. Although monocytes and their descendants (in particular, macrophages) have long been known to play an essential role in the degenerative process, only recently has their importance in positively influencing regeneration been recognized. Monocytes are abundant during nerve degeneration and regeneration and modulate the sequence of cellular events which can determine the outcome of the healing process.

After inflammatory insult, macrophages that accumulate at the site of injury appear to be derived largely from circulating monocytes. Entry of monocytes into the distal site of an injured nerve is enabled through up-regulation and release of a major monocyte chemokine, monocyte chemoattractant protein (CCL2) by SCs, which reaches its maximum 1 day after injury. Besides CCL2, the CX3CR1 ligand (fractalkine) can also recruit monocytes through the CX3CR1 receptor. In rats, two major subsets of monocytes have been identified. These two subtypes of monocytes can be recruited to injured tissues, where they can subsequently differentiate into classically activated (M1) or alternatively activated (M2) macrophages. These two phenotypes of macrophages represent a simplistic discrete depiction of a continuous spectrum between two activation states. Generally, M1 macrophages produce proinflammatory cytokines as well as high levels of oxidative metabolites, and M2 macrophages make the environment supportive for tissue repair by producing antiinflammatory cytokines that facilitate matrix remodeling and angiogenesis.

The plasticity of monocytes/macrophages makes them an attractive target for modulation in the context of nerve repair. A prior short-term study demonstrated that direct modulation of macrophages toward a prohealing phenotype, using interleukin 4 (IL-4), results in an increase in SC recruitment and axonal growth. The premise herein is that preferential recruitment of anti-inflammatory reparative monocytes to the site of injury will more effectively bias the immune microenvironment toward a prohealing response and in turn set off a regenerative biochemical cascade involving SCs and neuronal processes that leads to improved repair. Since CX3CR1 receptor is mainly expressed on antiinflammatory reparative monocytes, exogenous fractalkine, the ligand for CX3CR1, can be used to preferentially recruit these monocytes to the site of nerve injury and thus increase the subsequent ratio of prohealing to proinflammatory macrophages during the regeneration process.

Thus an immunomodulatory approach to stimulating nerve repair in a nerve-guidance scaffold was used to explore the regenerative effect of reparative monocyte recruitment. Early modulation of the immune environment at the injury site via fractalkine delivery resulted in a dramatic increase in regeneration as evident from histological and electrophysiological analyses. This study suggests that biasing the infiltrating inflammatory/immune cellular milieu after injury toward a proregenerative population creates a permissive environment for repair. This approach is a shift from the current modes of clinical and laboratory methods for nerve repair, which potentially opens an alternative paradigm to stimulate endogenous peripheral nerve repair.

Link: https://doi.org/10.1073/pnas.1705757114

There is every reason to believe that selective destruction of senescent cells in older individuals should improve heart health, lowering the risk of cardiovascular disease and dysfunction. Researchers who demonstrated 25% median life extension in mice engineered to lack senescent cells found improvements in a number of measures of cardiac health. There is a good deal of evidence for senescent cells to reduce stem cell activity and tissue regenerative capacity, perhaps through chronic inflammation and other consequences of changes in immune cell behavior. Senescent cells spur fibrosis, which disrupts small scale tissue structure with the formation of scar-like tissue, and the growth of fibrosis is important in heart tissue aging. And so on, through much of the list of problems in cell and tissue function known to be caused by the presence of senescent cells. In the paper I'll point out here, researchers focus specifically on oxidative signaling and rising levels of oxidative stress in the heart, and how senescent cells might be involved in this facet of heart aging.

Oxidative theories of aging were among the first such views of the causes of aging to be established in the modern era of cellular biochemistry. The original, simple theories that postulated aging as directly driven by oxidative damage to important molecules have since been put to one side in favor of more nuanced views. The failure of antioxidants, when applied generally, to slow aging is considered to disprove the simple view of aging as cellular damage that scales as the presence of oxidative molecules increases with age. It is certainly true that the oxidative molecules and signatures of harmful oxidative modification of vital proteins do increase with age, however. Equally it is also true that oxidative molecules and oxidative damage are used as signals in beneficial processes, such as the response to exercise - antioxidants can actually cause harm by interfering there. In short, oxidative metabolism is a complex, balanced process; that it is disrupted and runs awry with aging doesn't necessarily make it a cause rather than a consequence.

Over the years, attention has focused upon mitochondria, the power plants of the cell, as the prime source of oxidative molecules and possibly the prime source of disruption in oxidative metabolism in old tissues. Modestly increasing or reducing the flux of oxidative molecules generated by these organelles can improve health in short-lived species - in one direction by causing less damage and in the other by spurring greater repair and maintenance activities. Severe disruption of correct mitochondrial function, something that is known to occur in aged tissues, can drive cells into a failure state that results in large quantities of oxidative molecules pumped out into the surrounding tissues. To close the circle, mitochondrial dysfunction goes hand in hand with cellular senescence, though the bigger picture of cause and consequence here is complicated and still incomplete.

Cardiac Cell Senescence and Redox Signaling

Among age-related diseases, cardiovascular disease has an impressive prevalence, considering that the remaining lifetime risk for cardiovascular disease is about 50% at the age of 40. Consistently, the pathophysiologic modifications that are observed in aging hearts and arteries interact with alterations that characterize atherosclerosis progression, concurring to the development of age-associated heart failure. This latter is due to a combined diastolic and systolic dysfunction, caused by cardiac hypertrophy, replacement fibrosis, and myocardial ischemia, even in the absence of atherosclerotic coronary disease.

Morphometric data acquired in the early 1990s suggested that the number of left ventricle cardiomyocytes declines progressively with aging. Consistently, investigators have documented that although cardiomyocyte turnover occurs postnatally, the rate of cardiomyocyte renewal declines as age advances. Intriguingly, while the same investigators have recently suggested that the total number of cardiomyocytes residing in the left ventricle does not change with aging, evidence of myocyte death has been shown to occur both in male primates and in humans. In these latter, cardiac troponin T levels increase with aging and can predict cardiovascular events and death in the general population. This finding is thought to be the consequence of the age-related reduction of expression or activity of proteins that are involved in cardioprotection, a condition that eventually leads to an increased susceptibility of cardiac myocytes to injury.

To understand the mechanisms leading to heart failure, we and other authors hypothesized that the reduced cardiomyocyte turnover observed in aging was a consequence of the reduced cardiac growth reserve. Several independent groups have shown that undifferentiated, primitive cells reside in mammalian hearts and are involved in cardioprotection against heart failure, possibly generating new myocytes. Conversely, different lines of evidence obtained in animal models of heart failure and in humans indicate that senescent and dysfunctional cardiac resident stem/progenitor cells (CS/PC) accumulate as a consequence of cardiac pathology. Furthermore, with organism aging, senescent primitive and differentiated cells accumulate in mammalian hearts.

Although the concept of cellular senescence was introduced more than 50 years ago, the debate around this programmed cellular behavior is still ongoing. Specifically, in relatively recent years, it has been shown that cell senescence may exert positive effects, by promoting tissue healing after injury and protecting young organisms from cancer. However, in line with the antagonistic pleiotropy theory of aging, these beneficial effects exerted by cell senescence in young animals may be also responsible for the occurrence of functional impairment and age-related pathologies. Consistently, "rejuvenation" strategies aimed at reducing the frequency of senescent cells in the organism or designed to modulate those pathways whose activation status is altered in cell senescence can restore cardiac function in aged and failing hearts. Finally, we should emphasize that, while it has been postulated that reactive oxygen species (ROS) play a primary role in the development of cell senescence, the molecular mechanisms responsible for the development and evolution of cellular senescence are still a matter of intense research.

For many years it was believed that ROS were produced in an unregulated manner as a byproduct of cellular metabolism. Moreover, their ability to cause damage to macromolecules was thought to be responsible for organism aging (also known as the mitochondrial free radical theory of aging, MFRTA). Consistently, several pieces of evidence have shown an age-dependent decrease in mitochondrial integrity, and a parallel increase in the level of oxidized DNA (including mitochondrial DNA). These alterations have led to the formulation of "the vicious cycle hypothesis of mitochondrial ROS generation," according to which the mitochondrial production of ROS would damage mitochondrial DNA (mtDNA) and lead to mitochondrial dysfunction, thus increasing ROS generation. However, discordant results have been obtained in more recent years, which have either supported or refuted the increased production of mitochondrial ROS with aging.

These seemingly contradictory results can be reconciled if we consider that ROS have a dual nature. In fact, on top of their ability to damage in non-specific fashion biological molecules, ROS can exert useful and beneficial effects, by regulating signaling pathways. According to current models, ROS generation is highly regulated, and therefore oxidative stress would arise from the loss of this architecture. Importantly, redox signaling is a crucial regulator of stem cell quiescence, self-renewal, and differentiation. Conversely, loss of controlled redox signaling (oxidative stress) can obliterate stem cell function and promote cell senescence of stem and differentiated cells, two conditions that have been associated with the progressive loss of tissue renewal and reparative reserve that characterize aging.

Cardiac stem cells are not immune from these pathological processes, becoming dysfunctional and unable to effectively repair cardiac damage with organism aging and pathology. mTOR signaling, which associates with cardiac stem cell senescence, may affect redox signaling at multiple levels, overloading the endoplasmic reticulum, and inhibiting both lysosomal function and autophagy. Therefore, innovative interventions aimed at restoring proper redox signaling in primitive cells have the potential to reverse or attenuate age-associated stem cell dysfunction.

The author of this paper is one of the more outspoken advocates in the research community when it comes to mTOR and rapamycin as a path to slowing the progression of aging. He keeps up quite the output of position papers, such as this one, which calls for immediate human trials of polypills made up of rapamycin and a brace of other drugs broadly used in treatment of age-related conditions, such as statins and metformin. I have to think that the evidence to date suggests this will be less effective than hoped, while still very plausibly being better than doing nothing, even considering the side-effects of the drugs involved. Effects in animal studies usually tend to be much more pronounced than effects in humans when it comes to slowing or preventing specific age-related diseases through pharmaceuticals.

If it was the only game in town, I'd be all for it, but there are far more effective ways forward towards the effective treatment of aging as a medical condition - approaches that aim at rejuvenation, not a mere slowing of aging. Still, I think the author here has the right general idea, in that the research community should move faster, the sooner plausible approaches are trialed the better, and that we should all pitch in to help, it is just that he is advocating a poor approach with a limited upside in comparison to other methodologies.

Inhibitors of mTOR, including clinically available rapalogs such as rapamycin (Sirolimus) and Everolimus, are gerosuppressants, which suppress cellular senescence. Rapamycin slows aging and extends life span in a variety of species from worm to mammals. Rapalogs can prevent age-related diseases, including cancer, atherosclerosis, obesity, neurodegeneration and retinopathy and potentially rejuvenate stem cells, immunity and metabolism. Here, I suggest how rapamycin can be combined with metformin, inhibitors of angiotensin II signaling (Losartan, Lisinopril), statins, propranolol, aspirin and a PDE5 inhibitor. Rational combinations of these drugs can maximize their anti-aging effects and decrease side effects.

At first, the discovery of anti-aging properties of rapamycin was met with skepticism because it challenged the dogma that aging is a decline driven by molecular damage caused by free radicals. By now, rapamycin has been proven to be an anti-aging drug. In contrast, anti-oxidants failed in clinical trials and the dogma was shattered. In the last decade, anti-aging effects of rapamycin have been confirmed. Anti-aging doses and schedules can be extrapolated from animal studies. Well-tolerated doses with minimal side effects can be deducted based on clinical use of rapalogs. So optimal anti-aging doses/schedules can be suggested. Given that rapamycin consistently extends maximal lifespan in mice, rapamycin will likely allow mankind to beat the current record of human longevity, which is 122 years. Yet, rapamycin will not extend life span as much as we might wish to. Now is the time for anti-aging drug combinations. For example, metformin is currently undergoing re-purposing as an anti-aging agent. Several other existing drugs can be re-purposed. Now we can design an anti-aging formula, using drugs available for human use.

Rapamycin (or another rapalog) should be a cornerstone of anti-aging combinations, given its universal anti-aging effect and the ability to delay almost all diseases of aging. Rapamycin and metformin: Both drugs extend lifespan in animals and have non- overlapping effects. In addition, they may, in theory, cancel possible metabolic side-effects of each other. Rapamycin and statins: Rapamycin promotes lipolysis increasing blood levels of fatty acids. This, in turn, increases levels of lipoproteins produced by the liver. Rapamycin-induced hyperlipidemia is benevolent and reversible. Still, statins are already used to prevent rapamycin-induced hyperlipidemia.

The 7-drug combination can be tested in mice, especially in mice on high fat diet and in cancer-prone mice. If started late in life, the experiments will take just several months to evaluate the effect on lifespan and cancer incidence as well as weight, blood pressure, glucose, insulin, triglycerides and leptin. In humans, the treatment program can be initiated regardless of any pre-clinical studies, because all 7 drugs are approved for human use and some of them such as aspirin and statin are widely used for disease prevention anyway. The only what is needed is to watch for side effects. Especially, heart rate, blood pressure, and glucose levels should be monitored.

The anti-aging formula is ready for human use. If one will wait until the life-extending effect will be shown in others, this individual will not be alive by the time of the result. Human clinical trials are needed to optimize the doses and schedules. However, unless we participate in clinical trials ourselves, we will not know how long participants will live because they are expected to outlive non-participants. If we want to live longer we should be participants in clinical trials. In the best scenario, this might allow us to live long enough to benefit from future discoveries of anti-aging remedies.

Link: https://doi.org/10.18632/oncotarget.18033

Genetic engineering is now a fast path to cures for inherited diseases, those in which the cause is mutation in a single gene. Remove or suppress the bad gene, and insert the fixed version. This sort of approach, suppressing or editing a few specific genes, is unlikely to be as broadly and directly effective for age-related conditions, however. This is because, as is the case for aging, the disease state is influenced by thousands of genes, but directly caused by none of them. There are arguments, such as the research noted here, that suggest we should expect discoveries such as myostatin knockout for muscle growth, or ASGR1 and ANGPTL4 for reduced cardiovascular disease risk, to be unusual in the magnitude of their effects. When in search of genetic alterations to beneficially change a disease state, we should expect to come up more or less empty handed most of the time.

Here I am focused on the standard approach of editing or manipulating expression of one gene or a few genes, and not on more extensive engineering projects such as the SENS rejuvenation research program that aims to copy altered forms of mitochondrial DNA into the cell nucleus as a backup to remove the consequences of age-related damage to mitochondrial DNA. Or consider the Oisin Biotechnologies delivery of programmable DNA machinery to destroy a cell depending on its state. There will no doubt prove to be a role for other ambitious and essentially genetic reworkings of the cell, ways to improve redundancy of components or expand capabilities to ensure greater resilience, such as by providing a package of new enzymes capable of tackling problematic forms of metabolic waste.

This is, however, a very different class of approach to the single gene editing efforts that make up most of the field at present. This may be an era of medicine dominated by genetics, but we must, I think, recognize where it is limited as well as where there is the greatest potential. To my mind I see far too much enthusiasm for the sort of focus on genetic personalized medicine and single gene manipulations put forward by the likes of Human Longevity, to pick a representative example, while there just isn't enough of a benefit in their approach to be excited by it.

In a provocative new perspective piece, researchers say that disease genes are spread uniformly across the genome, not clustered in specific molecular pathways, as has been thought. The gene activity of cells is so broadly networked that virtually any gene can influence disease, the researchers found. As a result, most of the heritability of diseases is due not to a handful of core genes, but to tiny contributions from vast numbers of peripheral genes that function outside disease pathways. Any given trait, it seems, is not controlled by a small set of genes. Instead, nearly every gene in the genome influences everything about us. The effects may be tiny, but they add up.

The researchers call their provocative new understanding of disease genes an "omnigenic model" to indicate that almost any gene can influence diseases and other complex traits. In any cell, there might be 50 to 100 core genes with direct effects on a given trait, as well as easily another 10,000 peripheral genes that are expressed in the same cell with indirect effects on that trait. Each of the peripheral genes has a small effect on the trait. But because those thousands of genes outnumber the core genes by orders of magnitude, most of the genetic variation related to diseases and other traits comes from the thousands of peripheral genes. So, ironically, the genes whose impact on disease is most indirect and small end up being responsible for most of the inheritance patterns of the disease.

Researchers have thought of genetically complex traits as conforming to a polygenic model, in which each gene has a direct effect on a trait, whether that trait is something like height or a disease. In earlier work on the genetics of height, researchers were surprised to find that essentially the entire genome influenced height. "It was really unintuitive to me. To be honest, I thought that it was probably wrong. I gradually started to realize that the data don't really fit the polygenic model. We started to think, 'If the whole genome is involved in a complex trait like height, then how does that work?'"

The polygenic model leads researchers to focus on the short list of core genes that function in molecular pathways known to impact diseases. So, therapeutic research typically means addressing those core genes. A common approach to gene discovery is to do larger and larger genome-wide association studies, but the team argues against this approach because the sample sizes are expensive and the thousands of peripheral genes uncovered are likely to have tiny, indirect effects. "After you get the first 100 hits, you've probably found most of the core genes you're going to get through genomewide association studies." Instead, the team recommends switching to deep sequencing the core genes to hunt down rare variants that might have bigger effects. "If this model is right, it's telling us something profound about how cells work that we don't really understand very well. And so maybe that puts us a little bit further away from using genome-wide association studies for therapeutics. But in terms of understanding how genetics encodes disease risk, it's really important."

Link: http://med.stanford.edu/news/all-news/2017/06/thousands-of-genes-influence-most-diseases.html

Aubrey de Grey of the SENS Research Foundation is the advocate and scientist at the center of a diverse network of people and organizations who, collectively, are changing the world when it comes to aging, medicine, and research. It wasn't so very long ago that the research community and its associated sources of funding were hostile towards any effort to consider the treatment of aging as a medical condition. Decades were lost to a scientific culture whose leading members wanted to distance themselves from "anti-aging" snake oil at any cost - including the sacrifice of any real possibility of progress. Change has come but slowly, and required outsiders such as de Grey to enter the research field and raise hell until the existing factions and establishments were forced to acknowledge the potential to extend healthy life and reverse the progression of age-related conditions. Younger researchers now benefit from a field in which they can build a better world, applying biotechnology to the causes of aging in order to alleviate this greatest cause of suffering and death. This field is no longer the poorly regarded backwater it once was, thanks to people like de Grey and his allies, but now one of the most exciting areas of modern life science research, the seed that will blossom into a vast and enormously beneficial industry in the years ahead.

Yet this is a transformation still in progress. The first battles have been won, the first rejuvenation therapies after the SENS vision of damage repair - those involving clearance of senescent cells - are well on their way to the clinic. But the majority of research programs and funding sources remain slow to change course. Funding for aging research remains minimal in comparison to funding for other areas of medicine. Where there is funding, it is still largely directed towards initiatives that cannot possibly do more than slightly slow aging, or merely patch over the symptoms of aging, as little attention is given to the cell and tissue damage that is the root cause of all age-related disease, dsyfunction, and death. Longevity science is a field in which the greatest challenge is not the discovery of great swathes of new information about aging, but rather to persuade the research community to make proper use of what is already known, and then fund that work sufficiently. All of the necessary classes of therapy needed for rejuvenation can be constructed based on the knowledge of twenty years ago; the development plans are set out in some detail. Yet all too much of the field remains focused on continued exploration of the details of aging as it operates in the absence of intervention.

This is where we come in. Our philanthropic support of organizations such as the Methuselah Foundation and SENS Research Foundation helps to move the research forward. Our investment in and support of startup companies working on SENS technologies helps to push meaningful therapies for aging closer towards the clinic. The growth and legitimacy of SENS and SENS-like rejuvenation research is something that our broader community has bootstrapped from an idea to its present state. We have succeeded to no small degree! There is much to do yet, however. Our ability to attract support to the most important lines of research and development has increased greatly in recent years, and will continue to soar as SENS approaches such as senescent cell clearance are proven out in trials and animal studies. Now is not the time to rest upon our laurels: so make a point to tell someone you know about the field of rejuvenation research, and that the promising therapies currently in development are the result of donations wisely made in past years. The more people who know today, the more supporters will join us in the years ahead, and this is far more a challenge of persuasion than a challenge of science at this stage.

Science Isn't The Reason That Humans Can't Live Forever

If humanity were to appoint a general in our war against aging, Aubrey de Grey would likely earn the honor. The British author and biomedical gerontologist has been on the front line for years, researching ways to free the world of age-related disease and, ultimately, extend human life indefinitely. From the SENS Research Foundation Research Center (SRF-RC) in Mountain View, CA, foundation scientists conduct proof-of-concept research with the goal of addressing the problems caused by aging. They focus on repairing damage to the body at the molecular level, and their work is helping advance the field of rejuvenation biotechnology.

SRF-RC teams are currently focusing on two equally complex-sounding research projects, one centered on allotopic expression (a way to bypass the harmful effects of age-caused mitochondrial mutations) and the other on telomerase-independent telomere elongation (a little-researched process by which some cancer cells overcome mortality). Either project could lead to major breakthroughs in anti-aging treatments, but as de Grey explains, the path to immortality doesn't just run through the science lab. While the research being conducted at the SRF-RC is far from simple, de Grey claims DNA mutations and cancer cells aren't the biggest hurdles to anti-aging breakthroughs: "The most difficult aspect of fighting age-related diseases is raising the money to actually fund the research." The nature of most science research is exploratory. Researchers don't know that what they're working on is going to yield the results they expect, and even if it does, turning basic research into income is no easy task. To support their work, most have to rely on funding from outside sources, such as government grants, educational institutions, or private companies.

"It's still an incredibly hard sell," de Grey claims. "We have very limited resources. We only have about 4 million dollars a year to spend, and so we spent it very judiciously." That money isn't going to just the two in-house projects, either. The SENS Research Foundation funds anti-aging research at institutions across the globe and provides grants and internships for students, so raising money to support those endeavors is key to continued success in its fight against aging. The benefits of ending the problem of aging would be tremendous. Not only would we be living longer, we'd be living healthier for longer.

Essential to raising money for anti-aging research is ensuring that those with the funds understands why it's worth the investment - a not-so-easy task given current misconceptions about aging. In 2015, eight major aging-focused organizations, released a report detailing what they call the many "notable gaps" that exist between expert perspectives on aging and the public's perception of the process. If the public isn't well informed on aging, it's even less knowledgeable about anti-aging. Fifty-eight percent of respondents in a 2013 Pew Research study said they had never even heard of radical life extension before. When asked if they would undergo treatments that would allow them to live to the age of 120 or older, the majority of those surveyed said they would not, and 51 percent thought such treatments would be "bad for society."

"There is still a huge amount of resistance to the logic that aging is bad for you and that it's a medical problem that needs to be addressed," explains de Grey. "It's really, really extraordinary to me that it's so hard to get this through to people, but that is the way it is. Aging is not mysterious. We understand it pretty well. It's not even a phenomenon of biology. It's more a phenomenon of physics. Any machine with moving parts is going to damage itself ... and the result is inevitably going to be that eventually the machine fails. It's the same for the human body as it is for a car, for example, and if we think about it that way, it becomes pretty easy to actually see what to do about it."

Researchers are these days feeling more confident in the identification of cytomegalovirus as a significant cause of immune system dysfunction in aging, as the conference report here illustrates. We might hope that this growing interest in cytomegalovirus in the context of aging will lead to more funding of means to repair the situation, aiming to restore some of the youthful capability to respond to pathogens and destroy potentially dangerous cells.

The immune system is an adaptable machine, but one with limits. In adults new immune cells are generated at a very slow pace in comparison to the overall count of such cells in circulation. This effectively produces what looks a lot like a limit on the number of immune cells. As the years pass, that limited population is increasingly taken over by endlessly duplicated memory cells specialized to cytomegalovirus. Uselessly specialized, as the immune system cannot effectively clear this virus from the body. Near everyone is infected by cytomegalovirus by the time old age is reached, but aside from its insidious long-term effects on the immune system, it causes no noticeable problems in the vast majority of people. Thus approaches to tackling it have not been given any great priority in the medical science community of past decades. When much of the immune system is overtaken by cytomegalovirus-specific cells, however, that leaves all too little room for cells capable of productively carrying out other functions, and the result is a failing immune system, characteristic of the old.

The best near-team approach to this problem is probably some form of selective destruction of the unwanted specialized immune cells, in order to free up capacity. That can be coupled with the generation of replacement immune cells from a patient cell sample, returned to the body to quickly make up the numbers. This is a very plausible goal, given the various trials and technology demonstrations of immune cell clearance for therapeutic purposes. The greatest challenge involved is to develop targeted cell destruction approaches that are safe and have minimal side effects for the patient, in comparison to the damaging pharmaceuticals used to date in human trials.

Nearly two decades ago, two key findings connected cytomegalovirus (CMV) with immune senescence. In 1999, researchers showed that CMV-positive and -negative humans exhibit dramatically different T cell subset ratios and that the effect seems to be increasing with aging. Around the same time, others described "memory inflation" driven by CMV in mice. Since then, numerous studies have been published investigating the associations between human, non-human primate or murine CMV with their respective hosts in the course of aging. The interest in the topic has been so sustained that it led to the establishment of CMV and Immunosenescence Workshops. This overview summarizes the state of the field before and the discussions at the 6th International CMV and Immunosenescence Workshop.

CMV, a member of beta-herpesvirus family, is the largest human virus. As is the case for other herpesviruses, following a brief acute infection period that elicits a typical CD8 T cell response, as well as CD4 and B cell responses, CMV establishes persistence that includes latency. Persistence/latency is established in reservoir cells that are distributed broadly across the organism. However, it is clear that the primary CMV infection is followed by a period of viral shedding and it remains unclear whether the virus, that is a master in immune evasion, ever really is truly latent in all its reservoirs or whether some cells produce it as a smoldering infection at low levels at most, if not all times. As a consequence, a large population of CMV-specific CD8, and to a lesser extent, CD4 T cells, is generated in response to cycles of viral reactivation (aptly called memory inflation). How exactly this memory inflation impacts the ability of an older immune system to function and provide defense against other infections is one of the key topics of interest.

The 6th International Workshop on CMV and Immunosenescence was organized with a primary goal to fill a gap identified at several recent meetings. Specifically, in addition to the topics reviewed above from the fifth workshop, it was felt that stronger attention must be focused on the biology of the virus itself and on its interactions with the host in the course of latency and reactivation. In fact, one of the greatest weaknesses of research into HCMV and immunosenescence comes from our inability to control and measure the state of viral activity. Another area of major interest is the impact of CMV on diseases on aging - whether and how the virus may be involved in modulating frequent age-related morbidities, in particular cardiovascular diseases (CVD), where there are strong epidemiological associations between CMV infection and morbidity and mortality from CVD.

Link: https://doi.org/10.1007/s11357-017-9984-8

This paper is an example of the further explorations of DNA methylation and aging presently taking place in the research community. DNA methylation is one of the epigenetic decorations to DNA that alter gene expression, and thus the pace at which specific proteins are generated in the cell. A few different epigenetic clocks have been discovered in recent years, patterns of change in DNA methylation levels that correlate well with biological age, in that people of a given chronological age who are more damaged and impacted by aging than their peers tend to have distinctively different DNA methylation patterns. Moving beyond the existing epigenetic clocks, researchers are now searching for more and better correlations, as well as specific mechanistic links with other cellular processes already known to change with age.

Alteration of the ribosome biogenesis and an overall protein synthesis rate decline have been observed to characterize aging process in many organisms, including humans. This decline could be an effect of the progressive deterioration in most cellular functions usually associated with aging, or it could be a concurrent factor in the process. If to date a general reduction of protein synthesis has been attributed to the decreased frequency of mRNA translation, current studies, reporting an involvement of epigenetic mechanisms in silencing a large fraction of the ribosomal RNA (rRNA) genes, with a consequent impairment of ribosomal DNA (rDNA) function, could lead to a new understanding of the phenomenon.

On the basis of this evidence, we investigated whether changes in the DNA methylation patterns of the rRNA gene promoter take place during the lifetime. DNA samples were extracted from whole blood collected from differently aged human individuals displaying different phenotypes according to cognitive, functional, and psychological parameters. We did not find a consistent statistically significant association between the methylation levels of the analyzed CpG sites with the age of the donor. On the other hand, although it is not associated with chronological aging, in middle/advanced-aged subjects the variability of CpG_5 methylation was found to be significantly correlated with both cognitive performances and survival in the 9-year follow-up period. This last result, which held multiple test correction, was further confirmed in the replication sample.

Our results seem to be particularly attractive, because they show a fine remodeling of the methylation profile associated with the biological aging rather than to the chronological age. The effects at molecular levels of the above association have to be clarified, but it is plausible to hypothesize that the decrease in the rRNA levels we observed late in life may be determined by the methylation of the CpG_5 site that in turn might be driven by multiple factors, including genetic variations, diet, environment, and the interindividual variation of the structure of rDNA cluster itself.

How could the methylation changes of peculiar CpG sites be functionally involved in the functional decline characterizing the aging process? If the epigenetic modification of functional sites may hamper ribosomal biogenesis, this may drastically reduce the cellular protein synthesis, being ultimately responsible of those multisystem deficits occurring over the lifetime. Thus, an interdependence seems to exist between rDNA promoter methylation and the aging process, and in particular with the aging associated decay, and these sites may represent an potential evolutionary conserved biomarker of the rate of the aging process.

Link: https://doi.org/10.1111/acel.12603

The progress in various approaches to gene therapy over the past decade has succeeded in reducing cost and increasing reliability. This has reached the point at which researchers can afford, from the point of view of both time and funding, to begin to combine gene therapies with other areas of medicine under development. In particular, reliable gene therapies targeting the controlling switches and dials of cell growth and regeneration should be a way to greatly improve the effectiveness of cell therapies and other, similar forms of regenerative medicine. The research reported below is a good example of the type, in which scientists combine a scaffold-based cell therapy with gene therapy to encourage local cells towards increased, controlled bone regrowth to replace severe fracture damage.

There are many methods of delivery for the introduction of therapeutic genes. The familiar use of viruses as a vector for the transfection of genes into a cell is just one class of approach - perhaps the most obvious one, given that viruses are in essence machines whose primary purpose is to place DNA into cells. But there are other approaches. Considered in the larger context, this diversity is a good thing, as greater competition and exploration always leads to a superior end result once all is said and done. The method used here is one of the pore-forming variations, in which one or another form of stimulus induces cells to open pores in the cell membrane and let in the DNA-bearing particles. In this case, the stimulus is physical, provided by cavitation of ultrasound-created microbubbles. It is worth bearing in mind that all of these methods have the potential to damage and kill cells, some more than others, and thus must be carefully calibrated. When it works, however, the results can be fairly impressive.

Injured Bones Reconstructed by Gene and Stem Cell Therapies

Investigators have successfully repaired severe limb fractures in laboratory animals with an innovative technique that cues bone to regrow its own tissue. If found to be safe and effective in humans, the pioneering method of combining ultrasound, stem cell and gene therapies could eventually replace grafting as a way to mend severely broken bones. "We are just at the beginning of a revolution in orthopedics. We're combining an engineering approach with a biological approach to advance regenerative engineering, which we believe is the future of medicine."

The new technique could provide a much-needed alternative to bone grafts. In their experiment, the investigators constructed a matrix of collagen, a protein the body uses to build bones, and implanted it in the gap between the two sides of a fractured leg bone in laboratory animals. This matrix recruited the fractured leg's stem cells into the gap over two weeks. To initiate the bone repair process, the team delivered a bone-inducing gene directly into the stem cells, using an ultrasound pulse and microbubbles that facilitated the entry of the gene into the cells. Eight weeks after the surgery, the bone gap was closed and the leg fracture was healed in all the laboratory animals that received the treatment. Tests showed that the bone grown in the gap was as strong as that produced by surgical bone grafts.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area.

Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.