Penn Engineers have pioneered a new way to visualize the smallest protein clusters, skirting the physical limitations of light-powered microscopes and opening new avenues for detecting the proteins implicated in diseases like Alzheimer’s and testing new treatments.
In a paper in Cell Systems, Lukasz Bugaj, Assistant Professor in Bioengineering, describes the creation of CluMPS, or Clusters Magnified by Phase Separation, a molecular tool that activates by forming conspicuous blobs in the presence of target protein clusters as small as just a few nanometers. In essence, CluMPS functions like an on/off switch that responds to the presence of clusters of the protein it is programmed to detect.
Normally, says Bugaj, detecting such clusters requires laborious techniques. “With CluMPS, you don’t need anything beyond the standard lab microscope.” The tool fuses with the target protein to form condensates orders of magnitude larger than the protein clusters themselves that resemble the colorful blobs in a lava lamp. “We think the simplicity of the approach is one of its main benefits,” says Bugaj. “You don’t need specialized skills or equipment to quickly see whether there are small clusters in your cells.”
In the human body, the lungs and their vasculature can be likened to a building with an intricate plumbing system. The lungs’ blood vessels are the pipes essential for transporting blood and nutrients for oxygen delivery and carbon dioxide removal. Much like how pipes can get rusty or clogged, disrupting normal water flow, damage from respiratory viruses, like SARS-CoV-2 or influenza, can interfere with this “plumbing system.”
In a recent study, researchers looked at the critical role of vascular endothelial cells in lung repair. Their work, published in Science Translational Medicine, was led by Andrew Vaughan of the University of Pennsylvania’s School of Veterinary Medicine and shows that, by using techniques that deliver vascular endothelial growth factor alpha (VEGFA) via lipid nanoparticles (LNPs), that they were able to greatly enhance modes of repair for these damaged blood vessels, much like how plumbers patch sections of broken pipes and add new ones.
“While our lab and others have previously shown that endothelial cells are among the unsung heroes in repairing the lungs after viral infections like the flu, this tells us more about the story and sheds light on the molecular mechanisms at play,” says Vaughan, assistant professor of biomedical sciences at Penn Vet. “Here we’ve identified and isolated pathways involved in repairing this tissue, delivered mRNA to endothelial cells, and consequently observed enhanced recovery of the damaged tissue. These findings hint at a more efficient way to promote lung recovery after diseases like COVID-19.”
They found VEGFA’s involvement in this recovery, while building on work in which they used single cell RNA sequencing to identify transforming growth factor beta receptor 2 (TGFBR2) as a major signaling pathway. The researchers saw that when TGFBR2 was missing it stopped the activation of VEGFA. This lack of signal made the blood vessel cells less able to multiply and renew themselves, which is vital for the exchange of oxygen and carbon dioxide in the tiny air sacs of the lungs.
“We’d known there was a link between these two pathways, but this motivated us to see if delivering VEGFA mRNA into endothelial cells could improve lung recovery after disease-related injury,” says first author Gan Zhao, a postdoctoral researcher in the Vaughan Lab.
“LNPs have been great for vaccine delivery and have proven incredibly effective delivery vehicles for genetic information. But the challenge here was to get the LNPs into the bloodstream without them heading to the liver, which is where they tend to congregate as its porous structure lends favor to substances passing from the blood into hepatic cells for filtration,” says Mitchell, an associate professor of bioengineering at Penn Engineering and a coauthor of the paper. “So, we had to devise a way to specifically target the endothelial cells in the lungs.”
Lulu Xue, a postdoctoral researcher in the Mitchell Lab and a co-first author of the paper, explains that they engineered the LNP to have an affinity for lung endothelial cells, this is known as extra hepatic delivery, going beyond the liver.
The development of any type of second cancer following CAR T cell therapy is a rare occurrence, as found in an analysis of more than 400 patients treated at Penn Medicine, researchers from the Perelman School of Medicine at the University of Pennsylvania reported today in Nature Medicine. The team also described a single case of an incidental T cell lymphoma that did not express the CAR gene and was found in the lymph node of a patient who developed a secondary lung tumor following CAR T cell therapy.
CAR T cell therapy, a personalized form of immunotherapy in which each patient’s T cells are modified to target and kill their cancer cells, was pioneered at Penn. More than 30,000 patients with blood cancers in the United States—many of whom had few, if any, remaining treatment options available—have been treated with CAR T cell therapy since the first such therapy was approved in 2017. Some of the earliest patients treated in clinical trials have gone on to experience long-lasting remissions of a decade or more.
Secondary cancers, including T cell lymphomas, are a known, rare risk of several types of cancer treatment, including chemotherapy, radiation, and stem cell transplant. CAR T cell therapy is currently only approved to treat blood cancers that have relapsed or stopped responding to treatment, so patients who receive CAR T cell therapies have already received multiple other types of treatment and are facing dire prognoses.
In November 2023, the FDA announced an investigation into several reported cases of secondary T cell malignancies, including CAR-positive lymphoma, in patients who previously received CAR T cell therapy products. In January 2024, the FDA began requiring drugmakers to add a safety label warning to CAR T cell products. While the FDA review is still ongoing, it remains unclear whether the secondary T cell malignancies were caused by CAR T cell therapy.
As a leader in CAR T cell therapy, Penn has longstanding, clearly established protocols to monitor each patient both during and after treatment – including follow-up for 15 years after infusion – and participates in national reporting requirements and databases that track outcomes data from all cell therapy and bone marrow transplants.
“When this case was identified, we did a detailed analysis and concluded the T cell lymphoma was not related to the CAR T cell therapy. As the news of other cases came to light, we knew we should go deeper, to comb through our own data to better understand and help define the risk of any type of secondary cancer in patients who have received CAR T cell products,” said senior author Marco Ruella, MD, an assistant professor of Hematology-Oncology and Scientific Director of the Lymphoma Program. “What we found was very encouraging and reinforces the overall safety profile for this type of personalized cell therapy.”
Autoimmune disorders are among the most prevalent chronic diseases across the globe, affecting approximately 5-7% of the world’s population. Emerging treatments for autoimmune disorders focus on “adoptive cell therapies,” or those using cells from a patient’s own body to achieve immunosuppression. These therapeutic cells are recognized by the patient’s body as ‘self,’ therefore limiting side effects, and are specifically engineered to localize the intended therapeutic effect.
In treating autoimmune diseases, current adoptive cell therapies have largely centered around the regulatory T cell (Treg), which is defined by the expression of the Forkhead box protein 3, orFoxp3. Although Tregs offer great potential, using them for therapeutic purposes remains a major challenge. In particular, current delivery methods result in inefficient engineering of T cells.
Tregs only compose approximately 5-10% of circulating peripheral blood mononuclear cells. Furthermore, Tregs lack more specific surface markers that differentiate them from other T cell populations. These hurdles make it difficult to harvest, purify and grow Tregs to therapeutically relevant numbers. Although there are additional tissue-resident Tregs in non-lymphoid organs such as in skeletal muscle and visceral adipose tissue, these Tregs are severely inaccessible and low in number.
“The major challenges associated with ex vivo (outside the body) cell engineering are efficiency, toxicity, and scale-up: our mRNA lipid nanoparticles (mRNA LNPs) allow us to overcome all of these issues,” says Mitchell. “Our work’s novelty comes from three major components: first, the use of mRNA, which allows for the generation of transient immunosuppressive cells; second, the use of LNPs, which allow for effective delivery of mRNA and efficient cell engineering; and last, the ex vivo engineering of primary human T cells for autoimmune diseases, offering the most direct pipeline for clinical translation of this therapy from bench to bedside.”
“To our knowledge, this is one of the first mRNA LNP platforms that has been used to engineer T cells for autoimmune therapies,” he continues. “Broadly, this platform can be used to engineer adoptive cell therapies for specific autoimmune diseases and can potentially be used to create therapeutic avenues for allergies, organ transplantation and beyond.”
Delivering the Foxp3 protein to T cells has been difficult because proteins do not readily cross the cell membrane. “The mRNA encodes for Foxp3 protein, which is a transcription factor that makes the T cells immunosuppressive rather than active,” explains first author Ajay Thatte, a doctoral student in Bioengineering and NSF Fellow in the Mitchell Lab. “These engineered T cells can suppress effector T cell function, which is important as T cell hyperactivity is a common phenotype in autoimmune diseases.”
The sting of a toothache or the discovery of a cavity is a universal dread. Dental caries, more commonly known as tooth decay, is an insidious adversary, taking a toll on millions of mouths worldwide. Caries can lead to pain, tooth loss, infection, and, in severe cases, even death.
While fluoride-based treatments have long been the gold standard in dentistry, this singular approach is now dated and has limited effect. Current treatments do not sufficiently control biofilm—the main culprit behind dental caries—and prevent enamel demineralization at the same time. This dual dilemma becomes particularly pronounced in high-risk populations where the onset of the disease can be both rapid and severe.
“Traditional treatments often come short in managing the complex biofilm environment in the mouth,” Koo, senior co-author on the study, says. “Our combined treatment not only amplifies the effectiveness of each agent but does so with a lower dosage, hinting at a potentially revolutionary method for caries prevention in high-risk individuals.”
David Cormode is an associate professor of radiology and bioengineering with appointments in Penn’s Perelman School of Medicine and School of Engineering and Applied Science.
Other authors are Yue Huang, Nil Kanatha Pandey, Shrey Shah, and Jessica C. Hsu of Penn’s Perelman School of Medicine; Yuan Liu, Aurea Simon-Soro, Zhi Ren, Zhenting Xiaang, Dongyeop Kim, Tatsuro Ito, Min Jun Oh, and Yong Li of Penn’s School of Dental Medicine; Paul. J Smeets, Sarah Boyer, Xingchen Zhao, and Derk Joester of Northwestern University; and Domenick T. Zero of Indiana University.
The work was supported by the National Institute of Health (grants R01-DE025848 and TL1TR001423 and awards S10OD026871 and R90DE031532) and the National Science Foundation (awards ECCS-2025633 and DMR-1720139).
Breaking the code of the immune system could provide a new fundamental way of understanding, treating, and preventing every type of disease. Penn Medicine is investing in key discoveries about immunity and immune system function, and building infrastructure, to make that bold idea a reality.
This grandfather lives with primary progressive multiple sclerosis (MS), an autoimmune disorder that he controls with a medicine that depletes his body of the type of immune cells that make antibodies. So while he has completed his COVID-19 vaccine course, his immune system function isn’t very strong—and the invitation has arrived at a time when COVID-19 is still spreading rapidly.
You can imagine the scene as an older gentleman lifts a thick, creamy envelope from his mailbox, seeing his own name written in richly scripted lettering. He beams with pride and gratitude at the sight of his granddaughter’s wedding invitation. Yet his next thought is a sober and serious one. Would he be taking his life in his hands by attending the ceremony?
“In the past, all we could do was [measure] the antibody response,” says Amit Bar-Or, the Melissa and Paul Anderson President’s Distinguished Professor in Neurology at the Perelman School of Medicine, and chief of the Multiple Sclerosis division. “If that person didn’t have a good antibody response, which is likely because of the treatment they’re on, we’d shrug our shoulders and say, ‘Maybe you shouldn’t go because we don’t know if you’re protected.’”
Today, though, Bar-Or can take a deeper dive into his patients’ individual immune systems to give them far more nuanced recommendations. A clinical test for immune cells produced in response to the COVID-19 vaccine or to the SARS-CoV-2 virus itself—not just antibodies—was one of the first applied clinical initiatives of a major new Immune Health® project at Penn Medicine. Doctors were able to order this test and receive actionable answers through the Penn Medicine electronic health record for patients like the grandfather with MS.
“With a simple test and an algorithm we can have a very different discussion,” Bar-Or says. A test result showing low T cells, for instance, would tell Bar-Or his patient may get a meaningful jolt in immunity from a vaccine booster, while low antibody levels would suggest passive antibody therapy is more helpful. Or, the test might show his body is already well primed to protect him, making it reasonably safe to attend the wedding.
This COVID-19 immunity test is only the beginning.
Physicians and scientists at Penn Medicine are imagining a future where patients can get a precise picture of their immune systems’ activity to guide treatment decisions. They are working to bring the idea of Immune Health to life as a new area of medicine. In labs, in complex data models, and in the clinic, they are beginning to make sense out of the depth and breadth of the immune system’s millions of as-yet-undeciphered signals to improve health and treat illnesses of all types.
Penn Medicine registered the trademark for the term “Immune Health” in recognition of the potential impact of this research area and its likelihood to draw non-academic partners as collaborators in its growth. Today, at the south end of Penn’s medical campus, seven stories of research space are being added atop an office building at 3600 Civic Center Blvd., including three floors dedicated to Immune Health, autoimmunity, and immunology research.
The concept behind the whole project, says E. John Wherry, director of Penn Medicine’s Institute for Immunology and Immune Health (I3H), “is to listen to the immune system, to profile the immune system, and use those individual patient immune fingerprints to diagnose and treat diseases as diverse as immune-related diseases, cancer, cardiovascular disease, Alzheimer’s, and many others.”
The challenge is vast. Each person’s immune system is far more complex than antibodies and T cells alone. The immune system is made of multiple interwoven layers of complex defenders—from our skin and mucous membranes to microscopic memory B cells that never forget a childhood infection—meant to fortify our bodies from germs and disease. It is a sophisticated system that learns and adapts over our lifetimes in numerous ways, and it also falters and fails in some ways we understand and others that remain mysterious. And each person’s intricate internal battlefield is in some way unique.
The immune system is not just a set of defensive barricades, either. It’s also a potential source of deep insight about a person’s physiological functioning and responses to medical treatments.
“The immune system is sensing and keeping track of basically all tissues and all cells in our body all the time,” Wherry says. “It is surveying the body trying to clean up any invaders and restore homeostasis by maintaining good health.”
“Our goal is to essentially break the code of the immune system,” says Jonathan Epstein, executive vice dean of the Perelman School of Medicine and chief scientific officer at Penn Medicine. “By doing so, we believe we will be able to determine your state of health and your response to therapies in essentially every human disease.”
Let’s say you typically eat eggs for breakfast but were running late and ate cereal. As you crunched on a spoonful of Raisin Bran, other contextual similarities remained: You ate at the same table, at the same time, preparing to go to the same job. When someone asks later what you had for breakfast, you incorrectly remember eating eggs.
This would be a real-world example of a false memory. But what happens in your brain before recalling eggs, compared to what would happen if you correctly recalled cereal?
In a paper published in Proceedings of the National Academy of Sciences, University of Pennsylvania neuroscientists show for the first time that electrical signals in the human hippocampus differ immediately before recollection of true and false memories. They also found that low-frequency activity in the hippocampus decreases as a function of contextual similarity between a falsely recalled word and the target word.
“Whereas prior studies established the role of the hippocampus in event memory, we did not know that electrical signals generated in this region would distinguish the imminent recall of true from false memories,” says psychology professor Michael Jacob Kahana, director of the Computational Memory Lab and the study’s senior author. He says this shows that the hippocampus stores information about an item with the context in which it was presented.
Researchers also found that, relative to correct recalls, the brain exhibited lower theta and high-frequency oscillations and higher alpha/beta oscillations ahead of false memories. The findings came from recording neural activity in epilepsy patients who were already undergoing invasive monitoring to pinpoint the source of their seizures.
Noa Herz, lead author and a postdoctoral fellow in Kahana’s lab at the time of the research, explains that the monitoring was done through intracranial electrodes, the methodology researchers wanted to use for this study. She says that, compared to scalp electrodes, this method “allowed us to more precisely, and directly, measure the neural signals that were generated in deep brain structures, so the activity we are getting is much more localized.”
Michael Kahana is the Edmund J. and Louise W. Kahn Term Professor of Psychology in the School of Arts & Sciences and director of the Computational Memory Lab at the University of Pennsylvania. He is a member of the Penn Bioengineering Graduate Group.
In just two years since CiPD was founded, the outcomes of this newly conceived research partnership have proven its value: microrobots that clean teeth for people with limited mobility, a completely new understanding of bacterial physics in tooth decay, enzymes from plant chloroplasts that degrade plaque, promising futures for lipid nanoparticles in oral cancer treatment and new techniques and materials to restore nerves in facial reconstructive surgery.
In addition, CiPD is training the next generation of dentists, scientists and engineers through an NIH/NIDCR-sponsored postdoctoral training program as well as fellowships from industry.
The two urge “the academic community to adopt a coordinated approach uniting dental medicine and engineering to support research, training and entrepreneurship to address unmet needs and spur oral health care innovations.”
In recent years, cancer researchers have hailed the arrival of chimeric antigen receptor T cell (CAR T) therapy, which has delivered promising results, transforming the fight against various forms of cancer. The process involves modifying patients’ T-cells to target cancer cells, resulting in remarkable success rates for previously intractable forms of cancer.
Six CAR T cell therapies have secured FDA approval, and several more are in the pipeline. However, these therapies come with severe and potentially lethal side effects, namely cytokine release syndrome (CRS) and neurotoxicity. These drawbacks manifest as a range of symptoms—from high fever and vomiting to multiple organ failure and patient death—posing significant challenges to broader clinical application.
“Addressing CRS and neurotoxicity without compromising the therapeutic effectiveness of CAR T cells has been a complex challenge,” says Mitchell.
He says that unwanted interactions between CAR T and immune cells called macrophages drive the overactivation of macrophages, which in turn result in the release of toxic cytokines that lead to CRS and neurotoxicity.
“Controlling CAR T-macrophage interactions in vivo is difficult,” Mitchell says. “So, our study introduces a materials engineering-based strategy that involves incorporating a sugar molecule onto the surface of CAR T cells. These sugars are then used as a reactive handle to create a biomaterial coating around these cells directly in the body, which acts as a ‘suit of armor,’ preventing dangerous interactions with macrophages.”
First author Ningqiang Gong, a postdoctoral researcher in the Mitchell Lab, elaborates on the technique, “We attached this sugar molecule to the CAR T cells using metabolic labeling. This modification enables the CAR T cells to attack cancer cells without any hindrance.”
“When symptoms of CRS begin to manifest, we introduce another molecule—polyethylene glycol (PEG)—to create the suit of armor, which effectively blocks dangerous interactions between these engineered T cells, macrophages, and the tumor cells themselves,” Gong says.
For most of modern medicine, cancer drugs have been developed the same way: by designing molecules to treat diseased cells. With the advent of immunotherapy, that changed. For the first time, scientists engineered patients’ own immune systems to recognize and attack diseased cells.
One of the best examples of this pioneering type of medicine is CAR T cell therapy. Invented in the Perelman School of Medicine by Carl June, the Richard W. Vague Professor in Immunotherapy, CAR T cell therapy works by collecting T cells from a patient, modifying those cells in the lab so that they are designed to destroy cancerous cells, and reinfusing them into the patient. June’s research led to the first FDA approval for this type of therapy, in 2017. Six different CAR T cell therapies are now approved to treat various types of blood cancers. Carl June, at the flash mob celebration of the FDA approval of the CAR T cell therapy he developed, in August 2017. (Image: Courtesy of Penn Medicine Magazine)
CAR T cell therapy holds the potential to help millions more patients—if it can be successfully translated to other conditions. June and colleagues, including Daniel Baker, a fourth-year doctoral student in the Cell and Molecular Biology department, discuss this potential in a perspective published in Nature.
In the piece, June and Baker highlight other diseases that CAR T cell therapy could be effective.
“CAR T cell therapy has been remarkably successful for blood cancers like leukemias and lymphomas. There’s a lot of work happening here at Penn and elsewhere to push it to other blood cancers and to earlier stage disease, so patients don’t have to go through chemo first,” June says. “Another big priority is patients with solid tumors because they make up the vast majority of cancer patients. Beyond cancer, we’re seeing early signs that CAR T cell therapy could work in autoimmune diseases, like lupus.”
As for which diseases to pursue as for possible future treatment, June says, “essentially it boils down to two questions: Can we identify a population of cells that are bad? And can we target them specifically? Whether that’s asthma or chronic diseases or lupus, if you can find a bad population of cells and get rid of them, then CAR T cells could be therapeutic in that context.”
“What’s exciting is it’s not just theoretical at this point. There have been clinical reports in other autoimmune diseases, including myasthenia gravis and inflammatory myopathy,” Baker says. “But we are seeing early evidence that CAR T cell therapy will be successful beyond cancer. And it’s really opening the minds of people in the field to think about how else we could use CAR T. For example, there’s some pioneering work at Penn from the Epstein lab for heart failure. The idea is that you could use CAR T cells to get rid of fibrotic tissue after a cardiac injury, and potentially restore the damage following a heart attack.”
Baker adds, “there’s no question that over the last decade, CAR T cell therapy has revolutionized cancer. I’m hoping to play a role in bringing these next generation therapies to patients and make a real impact over the next decade. I think there’s potential for cell therapy to be a new pillar of medicine at large, and not just a new pillar of oncology.”