Identification and Targeting of Noncanonical Death Resistant Cells

SENS Research Foundation Research Center

Forever Healthy Foundation Fellowship in Rejuvenation Biotechnology

Principal Investigator: Tesfahun Admasu

When cells age, they lose their proliferative capacity and stop dividing in a phenomenon called senescence. Cellular senescence decreases the regenerative capacity of cells and tissues.

Throughout the aging process senescent cells accumulate and secrete a characteristic set of proteins, called a senescence-associated secretory phenotype (SASP). Although SASPs act as tumor suppressors and recruit immune cells to repair damage, they also mediate the deleterious effects of senescence to cause different pathologies, such as cancer, neurodegenerative diseases and diabetes. Furthermore, SASPs induce senescence in the surrounding cells (secondary senescence), which aggravates the effect.

Small molecules, called senolytics, eliminate senescent cells and reduce certain age-associated disorders.

Project Goals

This project seeks to test the hypothesis that secondary senescent cells are different from primary senescent cells and would therefore need a different set of senolytics to eradicate. In addition, the project will study the role of the different SASP components involved in the spreading of senescence, and test the hypothesis that intervening in SASP signaling could be therapeutically viable.

A Small Molecule Approach to Removal of Toxic Oxysterols as a Treatment For Atherosclerosis

SENS Research Foundation Research Center

Principal Investigator: Matthew O’Connor
Research TeamAmelia Anderson, Carolyn Barnes, Angielyn Campo, Anne Corwin, Sirish Narayanan

Many diseases of aging are driven in part by the accumulation of “junk inside cells:” stubborn, damaged waste products derived from the metabolic processes particular to specific cell types. The accumulation of these wastes disables the cell type in question and leads to their dysfunction; when, after decades of silent accrual, a critical number of these cells become dysfunctional, diseases of aging characteristic of that tissue erupt. For example, atherosclerotic lesions form when immune cells called macrophages take in 7-ketocholesterol (7-KC) and other damaged cholesterol byproducts in an effort to protect the arterial wall from their toxicity, only to ultimately fall prey to that same toxicity themselves. These macrophages – now dysfunctional “foam cells” – become immobilized in the arterial wall and spew off inflammatory molecules that in turn promote advanced atherosclerosis, heart attack, and stroke. In other organs, the accumulation of damaged molecules inside vulnerable cells drives Alzheimer’s and Parkinson’s diseases, as well as age-related macular degeneration.

Dr. O’Connor’s team have identified a family of small molecules that may be able to selectively remove toxic forms of cholesterol from early foam cells and other cells in the blood. If effective, these small molecules could serve as the basis for a groundbreaking therapy that would prevent and potentially reverse atherosclerosis and, possibly, heart failure.

Research Highlights:

A lead compound was identified following evaluation of data from human blood sample tests in conjunction with computer modeling to predict the likely behavior of rationally-designed molecules. Preliminary testing has indicated performance consistent with enhanced activity relative to the existing family of compounds: specifically, the candidate molecules exhibit selective targeting of toxic cholesterol byproducts, with significantly reduced affinity for native cholesterol. A patent application for this lead compound and others to be derived from it has now been submitted.

The team is now working to refine their original assay with the expectation that it will more accurately reflect the desired activity on toxic and native cholesterol, and also on an entirely different chemical approach to improved molecules derived from the original family. We are also working with a potential contract laboratory to test the absorption, circulation to tissues, and disposal of our lead candidate, and to perform toxicity assays. SRF has recently acquired a new robotic system to run the assay, which our in-house engineer, Anne Corwin, is now working to set up and program; the end result will be an increase in throughput that allows more rapid testing of more molecules.

Engineering New Mitochondrial Genes to Restore Mitochondrial Function (MitoSENS)

SENS Research Foundation Research Center

Principal Investigator: Amutha Boominathan
Research Team: Bhavna Dixit, Carter Hall, Caitlin Lewis, Matthew O’Connor, Martina Velichkovska

Mitochondria are the tiny cellular “power plants” in our cells, which take energy from our food and convert it into a form that can be used to power the cell’s energy-intensive processes. Like other power plants, they generate waste in the process – in this case, free radicals – which over time damage mitochondrial DNA. As a result, a small but rising number of our cells get taken over by such dysfunctional mitochondria as we age. These damaged cells in turn export toxic molecules to far-flung tissues, contributing to Parkinson’s disease, age-related muscle dysfunction, and other conditions.

The MitoSENS goal is to achieve a grand engineering solution to the problem of accumulation of cells with these mutation-bearing mitochondria: allotopic expression of functional mitochondrial genes. Allotopic expression involves placing “backup copies” of all of the protein-coding genes of the mitochondria in the cell’s nucleus. From this “safe harbor”, the copied genes can then direct the cell’s machinery to produce engineered versions of the missing mitochondrial proteins and deliver them to the mitochondria. With their full complement of proteins restored, mitochondria can resume producing energy normally, despite lacking the genes to produce them on their own.

Research Highlights:

In 2016, the MitoSENS team achieved a major breakthrough in successfully demonstrating efficient replacement of the missing mitochondrial ATP8 gene in cells from a human patient with an ATP8 mutation, restoring their ability to produce energy using the most efficient pathway.

After significant work to extend 2016’s breakthrough to other genes, the team discovered that an established method already widely used in biotechnology could also be applied to enable significantly more consistent production of allotopically-expressed protein.

To test this novel method more broadly, the MitoSENS group first briefly allotopically expressed each of the thirteen vulnerable mitochondrial genes via a transient loop of DNA located in the cytosol. Versions of the genes engineered the new way produced a great deal more RNA (the “working copies” of the gene that the cellular machinery uses to make protein) than the same genes engineered in the way that all previous investigators have used.

All thirteen of the genes engineered in this new way were able to produce actual protein, versus only a fraction of the conventionally-engineered genes. This milestone achievement is being prepared for publication in a scientific journal as of this writing, and tests are now underway to verify that all proteins thus expressed are properly incorporated into the mitochondria’s energy-production system.

The team has compared performance between ‘traditional’ and novel systems for producing allotopic ATP8 in cells derived from FVB mice. These mice bear a minor but significant mutation in ATP8 that causes functional problems, e.g., a tendency to poorly metabolize incoming blood sugar after a meal. The cells engineered using this novel method produced significantly more ATP8 protein than those engineered the conventional way – and it is important to note that in this experiment, the new genes were actually cemented into the nucleus and expressed from there, thus mimicking the goal for human MitoSENS therapies. The allotopically-expressed protein works as intended when using the improved system: it enters the mitochondria, incorporates properly into the energy-producing machinery, and significantly enhances these cells’ ability to survive when they are forced to rely on the mitochondria’s primary energy-generation mechanism.

Next, the MitoSENS team plans to demonstrate efficacy in living, breathing mice – specifically,  Maximally Modifiable Mice (MMM) from the SRF funded work at ASC. The new MMM-derived mouse model will have the allotopic ATP8 construct engineered into their nuclear genomes from conception, but will have mitochondria (and thus mitochondrial DNA) derived from FVB mice, with their mutant ATP8 gene. This work, in conjunction with behavioral studies to be performed in collaboration with the Brand lab at the Buck Institute, is expected to prove that the allotopic gene actually functions in vivo, restoring the mice’s ability to generate cellular energy efficiently.

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
Research Team: Elena Fulon

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 (the “senescence-associated secretory phenotype” (SASP)). The charge sheet against senescent cells has now expanded into a remarkable litany of the diseases of aging.

Multiple studies have now, on a more encouraging note, 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. But in theory, 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.

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

Research Highlights:

Dr. Campisi has found that about ten percent of senescent cells are resistant to being killed, even by fresh NK cells, suggesting that these resistant cells are the ones that escape immunosurveillance and accumulate in aging tissues. Her research team and other scientists have developed preliminary data suggesting mechanisms whereby senescent cells can make themselves invisible to NK cells, thus protecting themselves from destruction.

The Buck-SRF-RC collaboration is now seeking to drill further down into these questions and test possible means to intervene in the process. The Campisi lab is looking into further elaborating the biology of one of senescent cells’ two self-protective mechanisms, and also testing a potential role for another kind of immune cell (macrophages) in defending the body against senescent cell accumulation.

At the SRF-RC, we are currently perfecting the method of co-culturing NK and senescent cells and controlling the killing process, and will begin testing two potential therapeutic targets identified in the Campisi lab. 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 for the first time to observe any direct effects of aging on NK cell senolytic activity. The team is also developing an algorithm for the SRF-RC’s automated microscope imaging system to rapidly analyze stained plates of cells for quantitative analysis of senescent cell-killing ability — a job hitherto done by laborious human visual microscopy.

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