Therapy to Destroy Cells with Reactivated “Jumping Genes”

Roswell Park Comprehensive Cancer Center

Principal Investigator: Andrei Gudkov
Research Team: Marina Antoch, Amy Stablewski, Nick Neznanov, Lilya Novototskaya, Olga Leontieva

Nearly half of the mammalian genome is long and short interspersed virus-like repetitive elements (LINEs and SINEs), which spread through the process of retrotransposition. Numerous intracellular mechanisms exist to silence these elements, but unfortunately these mechanisms decline with age. Active copies of these elements thus cause DNA damage over time as they integrate into genes and disrupt their functions. Cells with many such dysfunctions can mimic viral infections and trigger the production of the pro-inflammatory substance interferon (IFN). Normally, such cells are removed by the immune system, but as the immune system itself ages, it becomes less effective at this type of cleanup. This leads to “inflamm-aging”, which damages tissues further.

Analysis of blind mole rats, which exhibit both a natural IFN-driven mechanism for eradicating cells with damaged DNA as well as exceptionally long lifespans, has yielded observations supporting the LINE/SINE hypothesis.

SRF is sponsoring a study to test the impact of the removal of damaged cells on healthspan and lifespan, via the creation of a special transgenic mouse at the Roswell Park Comprehensive Cancer Center.

Research Highlights:

This project began in the last quarter of 2019, and the Roswell Park team is now generating the transgenic mouse model. The group has already verified the transgene expression and activity in a cell culture model. Newborn mice are currently being screened for successful integration of the gene construct designed by the team. The final mouse model will allow the team to track the amount of IFN positive cells, and a lifespan study will begin later this year.

Enhancing Innate Immune Surveillance of Senescent Cells

Buck Institute for Research on Aging

Principal Investigator: Judith Campisi
Research Team: Abhijit Kale

SENS Research Foundation Research Center

Principal Investigator: Amit Sharma/Alexandra Stolzing
Research Team: Elena Fulton

When normal cells lose their ability to replicate, they become senescent cells. Over time, senescent cells accumulate in aging tissues, spewing off a cocktail of inflammatory and growth factors, as well as enzymes that break down surrounding tissue and cause inflammation. This cocktail is the “senescence-associated secretory phenotype” or SASP. Senescent cells – and the downstream impact of the SASP – are now implicated in a remarkable litany of the diseases of aging.

On a more encouraging note, multiple studies have now documented that senolytic drugs and gene therapies that destroy senescent cells exert sweeping rejuvenating effects in aging, both in laboratory animals and animal models of multiple diseases of aging. In theory, however, senolytic therapies shouldn’t be necessary. The body’s immune system is on continuous patrol against senescent cells: our natural killer (NK) cells recognize senescent cells as abnormal, bind to them, and release substances that trigger the senescent cells to self-destruct.

An SRF-donor-funded collaboration between Dr. Judith Campisi’s lab at the Buck Institute and the SRF Research Center seeks to discover why senescent cells accumulate with age, and what might we do to enhance immune surveillance and elimination of these cellular saboteurs?

Research Highlights:

The Campisi lab has recently published three papers describing the underlying mechanism of immune evasion by resistant senescent cells (Pereira et al., 2019, Munoz et al., 2019, and Kale et al., 2020). Dr. Campisi has found that a significant proportion of senescent cells manage to evade destruction, even by fresh NK cells. These ‘resistant’ cells escape immunosurveillance and accumulate in aging tissues. Senescent cells moreover shed decoy ligands binding to NK cell receptors; another aim of this work is to screen for more such ligands shed by senescent cells.

The Buck-SRF-RC collaboration is now seeking to drill further into the mechanism of senescent cell accumulation, and test interventions. At the SRF-RC, we are currently perfecting the method of co-culturing NK and senescent cells and controlling the killing process;  next, we will begin testing therapeutic interventions.

The SRF-RC scientists are also working for the first time with NK cells derived directly from aged human donors (rather than long-cultured lines of NK cells, or NK cells artificially “aged” by exposure to oxidative stress or extensive replication in culture, as has been done in the past). Using these cells will allow them to observe any direct effects of aging on NK cell senolytic activity.

Functional Neuron Replacement to Rejuvenate the Neocortex

Albert Einstein College of Medicine

Principal Investigator: Jean Hébert
Research TeamHiroko Nobuta, Joanna Krzyspiak, Alexander Quesada, Marta Gronska-Peski, Jayleecia Smith

Of all the challenges in cell therapy, replacement of neurons in the neocortex is both the most important (the brain being the seat of consciousness and identity) and perhaps the most formidable. Only recently have any researchers succeeded in integrating new neurons into this area of the brain. Moreover, the vast majority of transplanted cells in these cases have failed to survive, and the few survivors have achieved only limited function and integration into existing circuits.

Research Highlights:

SRF sponsors Dr. Jean Hébert’s work to advance an innovative strategy to address this challenge. Dr. Hébert’s team has transplanted neuronal precursors along with precursors of the blood vessels needed to nourish and enhance the survival and integration of new neurons. Initial analysis shows that this approach is successful.

However, as transplanting neurons throughout the entire aging neocortex would be extremely invasive, posing serious risk of injury to a tissue we cannot afford to damage, the team has engineered microglia (which, unlike neurons and their precursors, are highly mobile cells) to disperse widely from the site of transplant. These microglia can then be reprogrammed into cortical projection neurons at their destination.

Initial work on the transdifferentiation of microglia into neurons has been established. From there, the team will characterize the integration of the transplanted microglia-turned-neurons into host circuits of converted neurons derived from our engineered transplanted microglia. It has been demonstrated that depletion of microglia can enhance the process and facilitate good dispersion of newly transplanted microglia; Dr. Hébert’s team aims to determine whether depleting host microglia enhances these processes in different models.

Glucosepane Crosslinks and Undoing Age-Related Tissue Damage

This research program has successfully spun-out into a company! Visit the Revel Pharmaceuticals website for more information on their transformative approach to tissue cross-linking and vascular disease.

Yale University

Principal Investigator: David Spiegel
Research Team: Prof. Jason Crawford, Nam Kim, Venkata Sabbasani, Matthew Streeter

The long-lived collagen proteins that give structure to our arteries and other tissues are continuously exposed to blood sugar and other highly reactive molecules necessary for life. Occasionally, these sugar molecules will bind to tissue collagen by sheer chemistry, and if not quickly reversed these initial links will in turn bind adjacent strands of collagen, reducing the range of motion of the tissue like the legs of runners in a three-legged race. As a result, these tissues slowly stiffen with age, leading to rising systolic blood pressure, kidney damage, and increased risk of stroke and other damage to the brain.

It is currently thought that the single most common of these Advanced Glycation End-products (AGE) crosslinks is a molecule called glucosepane. A rejuvenation biotechnology that could cleave glucosepane crosslinks would allow bound arterial proteins to move freely again, maintaining and restoring the elasticity of the vessels and preventing the terrible effects of their age-related stiffening. SRF has provided funding to the Yale GlycoSENS group for several years now, in order to develop tools necessary for enabling the development of glucosepane-cleaving drugs.

Research Highlights:

The Yale group’s first major milestone – the first complete synthesis of glucosepane itself – was a sufficient tour de force to earn publication in the prestigious journal Science. In 2018, they were able to scale up this pilot-level method to produce glucosepane in quantities useful for industrial production, and also to synthesize three conformational variants (diastereomers) of glucosepane that may occur in vivo. They are now working on two more such variants. They have also used their synthetic glucosepane to develop glucosepane-targeting antibodies capable of labeling glucosepane in aging tissues, which they are now working up into a monoclonal antibody for mass production that will be compatible with human metabolism and will allow researchers to track the effects of potential glucosepane-cleaving drugs.

Finally, and most excitingly, they have now identified a lead candidate glucosepane-cleaving biocatalyst, and completed the evaluation of seven significant variants and their AGE-breaking mechanism. Today, work continues on synthesizing pentosinane (another common AGE crosslink) and additionally on the AGE-related compounds iso-imidazole and 2-aminoimidazole.

Maximally Modifiable Mouse

This project has recently concluded; further information will be published in the near future.

Applied StemCell, Inc.

Principal Investigator: Dr. Ruby Yanru Chen-Tsai

The CRISPR/Cas9 gene editing system has the ability to make precisely-targeted changes in the genetic sequence – a clear strength of the platform – but is limited in its lack of an obvious delivery mechanism. It’s reasonably easy to use CRISPR/Cas9 to modify individual cells, but there is no known (or clearly-foreseeable) way to deliver the system to human tissues in vivo while still retaining strong precision and without the risk of either silencing or mutating introduced or non-targeted genes.

Moreover, CRISPR/Cas9 is really only able to make relatively small changes to an existing gene. That’s great for correcting small but catastrophic mutations in existing genes, or rendering genes with a toxic gain-of-function inoperable — but it’s not much use for delivering the new genes that will be necessary to take advantage of it for delivery of rejuvenation biotechnologies.

The Maximally-Modifiable Mouse project aims to overcome this problem by allowing us to make use of a powerful gene insertion system (the integrase) used by phages — viruses that target bacteria as their hosts. The mycobacteriophage Bxb1 catalyzes precisely-targeted, one-way insertion of even very large genes into the host genome. Unfortunately, mammals lack the genetic “docking sites” that this integrase targets. To enable the development of models of diseases of aging and the rapid testing and eventual human delivery of rejuvenation biotechnologies, SENS Research Foundation has been funding Stanford gene therapy spinoff Applied StemCell (ASC) to create a line of Maximally-Modifiable Mice (MMM). The MMM will have two of the needed docking sites engineered directly into their genomes, which will then be ready for the insertion of new therapeutic transgenes at any time during the lifespan.

Research Highlights:

Currently, ASC is testing the ability of the system to deliver and integrate convenient test genes into the mice’s cells in vivo. They will then test the expression and functional protein production of those genes. We are especially excited by the potential to use this technology to both develop better models in which to test the allotopically-expressed mitochondrial genes that our in-house Mito team has been testing in cells, and to deliver those genes and actually test them in such mice.

Remediation of Aberrant Intracellular Tau

This project has recently concluded; further information will be published in the near future.

Buck Institute for Research on Aging

Principal Investigator: Dr. Julie Andersen
Research Team: Cyrene Arputhasamy, Manish Chamoli, Anand Rane

Aging brains accumulate aggregates composed of aberrant forms of the protein tau, both inside and outside of neurons. These aggregates are an important driver of “normal” age-related cognitive decline, as well as neurodegenerative diseases of aging like Parkinson’s (PD) and Alzheimer’s (AD) diseases. A number of rejuvenation biotechnologies targeting aberrant tau outside of cells are currently in clinical trials, with the idea that capturing these “seeds” of tau aggregates will interrupt its “infectious” cell-to-cell transmission. But to prevent the problem entirely – and eventually reverse it – requires new strategies to target aberrant tau inside of brain neurons.

Dr. Andersen’s team is being funded by SRF to test the idea that this tau accumulation may result from age-related dysfunction of the cellular “recycling centers” (lysosomes) due to the buildup of other kinds of intracellular aggregates, such as beta-amyloid, the other major damaged protein characteristic of the AD brain. If this is indeed the case, then the most effective remediation method for aberrant tau could entail using rejuvenation biotechnology to target these primary aggregates, thus allowing the cell to clear out its own burden of aberrant tau once lysosomal function is restored.

Neurons of patients with AD and other neurodegenerative aging diseases are often full of autophagosomes (APGs), the vesicles that form around targets for autophagy and in which they are dragged to the lysosome for degradation. This buildup is thought to result from a failure of lysosomal function, as the already-overburdened organelle refuses to take up any more cargo.

Research Highlights:

The Andersen lab has developed lines of human and rat neuronal cells that produce APGs with molecular tags that allow them to track the production and disappearance of APGs in neurons. They can use these tags to screen for compounds that increase the successful trafficking of APGs and their cargo to the lysosome. Compounds that pass this preliminary test will then be evaluated in neurons treated with small, soluble beta-amyloid aggregates, to see if these compounds will prevent or reverse the formation of insoluble aggregates of both beta-amyloid and tau.

Target Prioritization of Tissue Crosslinking

The Babraham Institute

Principal Investigator: Jonathan Clark
Research TeamMelanie Stammers

As discussed in the project summary for “Glucosepane Crosslinks and Undoing Age-Related Tissue Damage”, adventitious crosslinking of collagen and elastin contributes to the slow stiffening of our arteries and other tissues with age. Some of these crosslinks occur as a result of spontaneous chemistry (as in the case of AGE crosslinking). Others, meanwhile, are the unintended consequences of metabolic processes that modify collagen — either as “collateral damage,” or to help us endure short-term problems at the cost of contributing to the long-term burden of crosslinking damage that eventually compromises function.

Amidst all of this, the sheer number of crosslinks of a given kind may not be a good measure of how high a priority it ought to be for rejuvenation biotechnology: some crosslinks may have a disproportionate effect on tissue elasticity depending on where they occur in the protein strand, how tightly they bind, and how much they interfere with the body’s ability break down and renew the tissue.

Recognizing the importance of prioritizing our targets, SRF is funding a study of this question at the Babraham Institute in Cambridge. In this study, “normally”-aging, nondiabetic mice are administered labeled building blocks for protein, which are then incorporated into extracellular matrix proteins, whose turnover can then be studied. The study has thus far provided valuable insight into how crosslinks regulate tissue stiffness, as well as updated our understanding of the turnover times of collagen and elastin. The results are currently under review and will be published soon.

Research Highlights:

One early and surprising finding of this work was that tissue crosslinks of types one might assume permanent are in fact continuously being broken apart and re-forming under the stress and strain of normal activity. It is the balance between these reversible crosslinks and the truly irreversible ones that gives rise to many of the changing mechanical properties of aging collagen.

The Babraham group have also confirmed that the crosslink profile in each tissue is distinct from others (which is only partially explained by the tissue-specific mixture of elastin and collagen), and that both the mixture of proteins and the pattern of protein-specific crosslinks changes with age. Importantly, some of the crosslinks that have been reported by others to accumulate in aging tissues were not detected. They are also complementing chemical analysis of the tissues with functional tests of the effect of these crosslinks on tissue mechanical function. The team have furthermore detailed chemical profiles of skin and tendon, and run pilot studies on a number of other tissues. Additional work is ongoing in mice fed with various sugars to further elucidate the role of sugars on collagen turnover in vivo.

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