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Researchers at the Francis Crick Institute, UCL and MSD have identified a potential treatment target for a genetic type of epilepsy.
Developmental and epileptic encephalopathies are rare types of epilepsy which start in early childhood. One of the most common types of genetic epilepsy, CDKL5 deficiency disorder (CDD), causes seizures and impaired development. Children are currently treated with generic antiepileptic drugs, as there aren’t yet any disease-targeting medications for this disorder.
CDD involves losing the function of a gene producing the CDKL5 enzyme, which phosphorylates proteins, meaning it adds an extra phosphate molecule to alter their function. Until now, researchers have not been sure how genetic mutations in CDKL5 cause CDD.
Through their research, published today in Nature Communications, the researchers examined mice which lacked the Cdkl5 gene, and used a technique called phosphoproteomics to scan for proteins which are a target for the CDKL5 enzyme.
They identified a calcium channel, Cav2.3, as a target. Cav2.3 allows calcium to enter nerve cells, exciting the cell and allowing it to pass on electrical signals. This is needed for the nervous system to function properly, but too much calcium coming into cells can result in overexcitability and seizures.
The researchers then recorded from the calcium channels to see what was happening when they were not being phosphorylated by CDKL5. The channels were able to open, but were taking a lot longer to close, leading to larger and more prolonged currents flowing through them. This implies that CDKL5 is needed to limit calcium entry into cells.
The researchers also used nerve cells derived from stem cells taken from people with CDD, again observing that phosphorylation of Cav2.3 was reduced. This suggests that Cav2.3 function is potentially altered in humans as well as mice.

Mutations in Cav2.3 that enhance channel activity are already known to cause severe early onset epilepsy in a related condition called DEE69, which shares a lot of the same symptoms of CDD. These results suggest that Cav2.3 overactivity is a common feature of both disorders, and that inhibiting Cav2.3 could help with symptoms like seizures.
Sila Ultanir, Senior Group Leader of the Kinases and Brain Development Laboratory at the Crick, said: “At the moment, there’s a clear need for drugs which specifically target the biological nature of CDD. We’ve made a molecular link between CDKL5 and Cav2.3, mutations in which produce similar disorders. Inhibiting Cav2.3 could be a route for trials of future targeted treatments.”
Marisol Sampedro-Castañeda, postdoctoral researcher at the Crick, and first author, said: “Our research highlights for the first time a CDKL5 target with a link to neuronal excitability. There’s scattered evidence that this calcium channel could be involved in other types of epilepsy too, so we believe that Cav2.3 inhibitors could eventually be tested more widely.
“Our findings have implications for a large group of people, from the families affected by these conditions to researchers working in the rare epilepsy field.”
This research was funded by MSD and the Loulou Foundation, a private foundation dedicated to the development of therapeutics and eventual cures for CDD.
Jill Richardson, Executive Director and Head of Neuroscience Biology at MSD, said: “MSD is proud of this innovative research resulting from a collaboration with researchers at the Crick and UCL. We have collectively furthered our scientific understanding of the biological targets associated with the aetiologies of Developmental Epileptic Encephalopathies — an understanding we hope will contribute toward scientific progress in this important area of high, unmet medical need.”
The researchers are now working with Lario Therapeutics, a recently launched biotech company which is seeking to develop first-in-class CaV2.3 inhibitors as precision medicines to treat CDD and related neurodevelopmental syndromes.

In a breakthrough study published in the journal Cancer Cell, researchers have decoded critical genetic factors in intestinal metaplasia patients, shedding light on early signs and prevention strategies for stomach cancer — often a “ticking time bomb” as patients experience no or only mild symptoms in the early stages.
Intestinal metaplasia, which is a change in the cells of the mucous membrane lining the stomach that often stems from chronic gastritis and manifests with symptoms akin to acid reflux, is also a sinister link to stomach cancer. Individuals afflicted with intestinal metaplasia cells face a sixfold increased risk of succumbing to this lethal cancer.
In Singapore alone, stomach cancer ranks as the fourth leading cause of cancer deaths in men and the fifth among women, claiming 300 to 500 lives annually, largely due to late detection. Two thirds of stomach cancer patients are only diagnosed at an advanced stage.
Unveiling early indicators through collaborative breakthrough research
The longitudinal study, which represents the world’s largest genomic survey of patients with intestinal metaplasia, examines more than 1,100 tissue samples using powerful technologies such as single-cell RNA sequencing and spatial transcriptomics*. Based on this extensive survey, researchers identified 26 ‘driver genes’ that play a pivotal role in the transition to stomach cancer. This landmark finding provides a glimpse into the mechanisms governing the transformation and offers a critical window for early detection and targeted prevention.
“Advances in DNA sequencing have made it possible for us to uncover diverse cell populations within these stomach changes, hinting at their potential transformation into cancerous cells influenced by various factors. It’s akin to understanding the ticking mechanism of a time bomb,” explained Dr Huang Kie Kyon, co-first author and Senior Research Fellow with the Cancer & Stem Cell Biology Programme at Duke-NUS Medical School (Duke-NUS).
Professor Patrick Tan, Senior Vice-Dean for Research at Duke-NUS and a professor with the School’s Cancer & Stem Cell Biology Programme said: “The comprehensive dataset we’ve assembled provides unprecedented insights into the progression of cell changes in the stomach to cancer. By using both clinical information and genetic data from advanced molecular technologies, we can better predict which stomach conditions might turn into stomach cancer compared to using only clinical information. This can help in the development of new and more precise ways to prevent and stop stomach cancer.” Prof Tan is also a member of the Genome Institute of Singapore, Cancer Science Institute of Singapore, and Precision Health Research Singapore (PRECISE).

The multi-institutional effort by researchers from Duke-NUS, National University Hospital (NUH), National University of Singapore’s Yong Loo Lin School of Medicine (NUS Medicine) and Seoul National University Hospital reflects the strengths of Singapore’s multi-institutional cancer research ecosystem and its strong links with global partners. This study was supported by the Singapore Gastric Cancer Consortium (SGCC), a national translational research group comprising clinicians and scientists working in stomach cancer research from academic medical centres, universities, hospitals and research institutes across Singapore. The published work is derived from the prospective Gastric Cancer Epidemiology Programme cohort.
The study offers clues into whether intestinal metaplasia cells directly transform into stomach cancer. It was revealed that a subpopulation of intestinal stem-like cells in patients with intestinal metaplasia closely resembles early stomach cancer cells, pointing to a possible early origin and potential of its malignant future. This discovery highlights the importance of screening for intestinal metaplasia in managing stomach cancer risk.
Co-senior author Professor Jimmy So, Head & Senior Consultant, Division of General Surgery (Upper Gastrointestinal Surgery), NUH commented on the clinical implications: “This molecular roadmap of disease progression from intestinal metaplasia offers many translational opportunities. We can now explore more targeted surveillance for patients at highest risk, as well as anti-inflammatorial or antibiotic agents to intercept premalignant clones before they evolve into cancer, potentially leading to improved patient outcomes through early detection.” He is also a professor at the Department of Surgery, NUS Medicine.
More efficient and targeted preventive measures for populations
At the population level, the findings hold promise for refining screening strategies and allocating resources more effectively to intercept the development of gastric cancer in high-risk individuals, ultimately contributing to more efficient and targeted preventive measures. This is especially relevant in countries such as Singapore, where the incidence of stomach cancer is moderate compared to Japan and South Korea where stomach cancer incidence is high enough to warrant mass screening.
“Encouragingly, our results revealed that combining genomic data with clinical check-ups can make predictions about stomach cancer more accurate. This means we might use genetic tests, including simple and inexpensive blood tests, to identify people who are at a very high risk of getting stomach cancer. With this approach, we can divide people into groups based on their risk using either regular check-ups or these affordable blood tests. This helps to save resources by making sure those at the highest risk get the right tests and care they need,” added Professor Khay Guan Yeoh, Lead Principal Investigator of the Singapore Gastric Cancer Consortium and Senior Consultant in the Division of Gastroenterology & Hepatology, National University Hospital. Prof Yeoh is also the Kishore Mahbubani Professor in Medicine and Health Policy, Department of Medicine, NUS Medicine.
Senior author Associate Professor Chung Hyunsoo from Seoul National University Hospital stressed the clinical ramifications: “This breakthrough may refine screening protocols, enabling early interventions for high-risk patients, while sparing others unnecessary procedures.”
The research was funded by the Open Fund-Large Collaborative Grant that is supported by the National Research Foundation, Singapore and administered by the Singapore Ministry of Health’s National Medical Research Council. The team also received support from Singapore’s Ministry of Education, Cancer Science Institute of Singapore under the National University of Singapore and the Francis Crick Institute. In addition, the project could not have been made possible without the contributions of researchers from Tan Tock Seng Hospital, Singapore General Hospital, Changi General Hospital, Nihon University School of Medicine, Yonsei University Wonju College of Medicine and The Chinese University of Hong Kong.
* Single-cell RNA sequencing (scRNA-seq) is a powerful technology that allows scientists to study individual cells’ genetic material (RNA) one cell at a time. Spatial transcriptomics is another technique used to study gene expression in tissues but with an added dimension of spatial information. It allows scientists to see where specific genes are being expressed within tissues or organs, providing insight into the organisation and communication between cells in their actual location within the tissue.

Caffeine can have a negative impact on football players’ decision-making skills, new research shows.
A study by Staffordshire University and Shiraz University in Iran has found that while consuming caffeine before a game can improve the accuracy of football (soccer) passes, it can have an adverse effect on more tactical play involving a higher number of passes.
Dr Pooya Soltani, Senior Lecturer in Games Technology at Staffordshire University, explained: “Caffeine is one of the most popular dietary supplements which has been shown to provide benefits during exercise, including football. Studies have shown that caffeine can enhance attention, accuracy, and speed, as well as self-reported measures of energy and mood.
“However, the effects of caffeine on “higher” cognitive functions such as problem-solving and decision-making are often debated, so we decided to investigate this.”
Twelve young football players, aged between 16-17 years old, took part in a series of tasks to explore the impact of caffeine on decision-making and passing accuracy.
Participants performed five short (10m) and five long (30m) passes, as well as the Loughborough Soccer Passing Test which assesses skills including passing, dribbling, control, and decision-making. The researchers then used a computer task to measure decision-making in different gameplay scenarios, with participants asked to determine the best outcome of ten simulated pre-recorded events.
The participants completed the tasks once after taking 3 mg/kg body mass of caffeine and once after consuming similar amounts of placebo.

The footballers were 1.67% more accurate in short passes and 13.48% more accurate in long passes when they consumed caffeine compared to the placebo. However, participants’ decision-making was 7.14% lower and the Loughborough Soccer Passing Test scores were 3.49% lower when they consumed caffeine compared to the placebo.
Negar Jafari, from Shiraz University, said: “While the short pass accuracy remained consistent among almost all participants before and after caffeine consumption, the performance varied in the case of long passes. Moreover, most of the participants scored lower on decision-making and the Loughborough Soccer Passing Test after consuming caffeine. This may suggest that more complex tasks with a higher number of passes might negatively be affected by low doses of caffeine ingested one hour before playing.”
The researchers, however, are not suggesting that footballers should avoid caffeine completely and recommend further research into its effects on decision-making in the game.
“During a football match, players must process various cues such as opponents’ positions, team organisation, and time pressure. Decision-making in passing is particularly important, where a well-executed pass can create scoring opportunities,” Dr Soltani commented.
“Our findings show that this can be affected by caffeine intake and coaches may find these performance metrics useful to explore in training. A number of parameters can be involved — the dosage of caffeine relative to body weight, the frequency of caffeine intake and certain positions of the players or their playing styles. For example, a slight decrease in pass accuracy might be crucial for a midfielder but less impactful for a goalkeeper.”

For decades, scientists have noted an intriguing similarity between a deficiency in vitamin B12 – an essential nutrient that supports healthy development and functioning of the central nervous system (CNS) – and multiple sclerosis (MS), a chronic disease in which the body’s immune system attacks the CNS and which can produce neurodegeneration.
Both vitamin B12 (also known as cobalamin) deficiency and MS produce similar neurological symptoms, including numbness or tingling in hands and feet, vision loss, difficulty walking or speaking normally and cognitive dysfunction, such as problems with memory.
In a new study, published online December 8, 2023 in Cell Reports, researchers at Sanford Burnham Prebys, with collaborators elsewhere, describe a novel molecular link between vitamin B12 and MS that takes place in astrocytes – important non-neuronal glial cells in the brain.
The findings by senior study author Jerold Chun, M.D., Ph.D., professor and senior vice president of neuroscience drug discovery, and Yasuyuki Kihara, Ph.D., research associate professor and co-corresponding author, and colleagues suggest new ways to improve the treatment of MS through CNS-B12 supplementation.
“The shared molecular binding of the brain’s vitamin B12 carrier protein, known as transcobalamin 2 or TCN2, with the FDA-approved MS drug fingolimod provides a mechanistic link between B12 signaling and MS, towards reducing neuroinflammation and possibly neurodegeneration,” said Chun.   
“Augmenting brain B12 with fingolimod or potentially related molecules could enhance both current and future MS therapies.”
In their paper, the team at Sanford Burnham Prebys, with collaborators at University of Southern California, Juntendo University in Japan, Tokyo University of Pharmacy and Life Sciences and State University of New York, focused on the molecular functioning of FTY720 or fingolimod (Gilenya®), a sphingosine 1-phosphate (S1P) receptor modulator that suppresses distribution of T and B immune cells errantly attacking the brains of MS patients.

Working with an animal model of MS as well as human post-mortem brains, the researchers found that fingolimod suppresses neuroinflammation by functionally and physically regulating B12 communication pathways, specifically elevating a B12 receptor called CD320 needed to take up and use needed B12 when it is bound to TCN2, which distributes B12 throughout the body, including the CNS.  This known process was newly identified for its interactions with fingolimod within astrocytes. Importantly, the relationship was also observed in human MS brains.
Of particular note, the researchers reported that lower levels of CD320 or dietary B12 restriction worsened the disease course in an animal model of MS and reduced the therapeutic efficacy of fingolimod, which occurred through a mechanism in which fingolimod hitchhikes by binding to the TCN2-B12 complex, allowing delivery of all to the astrocytes via interactions with CD320, with component losses disrupting the process and worsening disease.
These new findings further support to the use of B12 supplementation – especially in terms of delivering the vitamin to astrocytes within the brain – while revealing that fingolimod can correct the impaired astrocyte-B12 pathway in people with MS. 
The scientists said it is possible that other S1P receptor modulators on the market, such as  Mayzent®, Zeposia® and Ponvory®, may access at least parts of this CNS mechanism.  The study supports B12 supplementation with S1P receptor modulators with the goal of improving drug efficacy for this class of medicines.
The study also opens new avenues on how the B12-TCN2-CD320 pathway is regulated by sphingolipids, specifically sphingosine, a naturally occurring and endogenous structural analog of fingolimod, toward improving future MS therapies, Chun said. 
“It supports creating brain-targeted B12 formulations. In the future, this mechanism might also extend to novel treatments of other neuroinflammatory and neurodegenerative conditions.”
Additional authors on the study include Deepa Jonnalagadda, Manisha Ray, Clayton Ellington and Richard Rivera, all at Sanford Burnham Prebys, Aran Groves, Sanford Burnham Prebys and UC San Diego; Arjun Saha, University of Southern California; Hyeon-Cheol Lee-Okada and Takehiko Yokomizo, Juntendo University; Tomomi Furihata, Tokyo University of Pharmacy and Life Sciences; Edward V. Quadros, SUNY-Downstate Medical Center.
The study was supported by a grant from the National Institute of Neurological Disorders and Stroke at the National Institutes of Health (R01NS103940), Novartis, MEXT/JSPS KAKENHI (18H02627, 19KK0199, 21H04798, 18K16246 and 21K08565 ). Further support was provided by the Uehara Memorial Foundation, Kanae Foundation for the Promotion of Medical Science, Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Human Frontier Science Program, plus the Medical Scientist Training Program and Pharmacology Training Grant at UC San Diego (T32GM007752).
The study’s DOI is 10.1016/j.celrep.2023

Many diseases are caused by proteins that have spun out of control. Unfortunately, so far, conventional drugs have been able to stop only a fraction of these troublemakers. A new class of drugs known as PROTACs holds great promise in pharmaceutical research. They mark proteins for targeted degradation by the cell’s own protein disposal system.
Many of today’s drugs are small, simple molecules. They usually work by regulating the activity of proteins involved in pathologically derailed processes—which is precisely what makes their development extremely complicated. Consequently, a highly adapted molecule has to be developed for each protein, to fit into the corresponding lock—the active center of the protein—like a high-security key. However, proteins actively involved in pathologically derailed processes make up only a fraction of the disease-related proteins. As a result, many proteins are still considered therapeutically “undruggable.”
Cancer protein Ras—not undruggable after all?
A majority of the undruggable proteins are compelling targets in cancer research. Perhaps the most prominent among them is the small Ras protein. A single small change in Ras is enough to irreversibly flip the switch for cell growth to “on”—with serious consequences: The cells proliferate rapidly and uncontrollably. Ras mutations occur in nearly a quarter of all tumors. In a groundbreaking study in 2013, a team of researchers led by Herbert Waldmann at MPI in Dortmund developed a new strategy to make Ras, which was previously considered undruggable, druggable: Instead of targeting Ras directly, the researchers used a specially developed molecule to thwart the auxiliary protein PDEd, manipulating the transport and thus the activity of Ras in the cell. However, the researchers did not manage to completely stop the cancer-driving activity of Ras.
Two-armed molecule marks cancer protein for degradation
Only two years after Waldmann’s work, American researchers developed a promising new class of drugs to eliminate pathological proteins: they are known as PROTACs (proteolysis-targeting chimeras). These compounds effectively hijack the body’s own protein waste removal system. The large molecule composed of two arms grabs the target protein on one side and the E3 ligase of the protein waste system on the other, which prompts the waste system to dispose of the pathological protein. “That’s an ingenious, truly outstanding scientific achievement,” says Waldmann. “Instead of inhibiting the target protein’s enzymatic activity in a complex process, PROTACs only need to bind to their target with high selectivity. Theoretically speaking, this principle is universally applicable to all proteins, including our Ras transporter PDEd, as we have successfully demonstrated in our current work,” he concludes.
Serendipitous discovery opens up new possibilities
The chemists Waldmann and Winter, along with their teams, created a new PROTAC consisting of the PDEd inhibitor they had developed. They linked the inhibitor to a well-studied molecule that is known to alert another degradation system which can also process larger cell components. “However, our screens have revealed that instead of activating what we call macroautophagy, our PROTAC activates the protein degradation system,” says Georg Winter. He goes on to explain: “What is particularly interesting is that our PROTAC binds a new ligase that was not accessible to the PROTAC strategy so far.”
Currently, there are practically only two E3 ligases that can be used as binding sites for PROTACs. However, there are more than 600 E3 ligases in our bodies. And some of them are only present in very specific tissues. “Tissue-specific ligases could be used to specifically control the site of drug activity,” says Waldmann, looking to the future. “Our rather fortuitous discovery allows for further biological and medicinal-chemical investigation into the ligases we have found. This could help expand the range of pharmaceutically usable PROTACs and, one day, enable the targeted degradation of proteins in specific tissues,” he concludes.

The electrical properties of cancer cells can provide information on their cancer type, state, and drug resistance. However, conventional platforms to measure these properties are complex and can only analyze a few cells. Researchers from the Tokyo University of Science have successfully developed a high-throughput device that measures the electrical properties of cancer cells through continuous flow electrorotation. The new platform offers a high degree of automation and can simultaneously analyze several cells.
Monitoring cancer cells effectively can help physicians with treatment and management, thus reducing cancer-related mortality. Can non-invasive technologies pave the way for improved monitoring to reduce cancer mortality rates? Diagnostic platforms that non-invasively measure the electrical properties of cancer cells offer promise in the early detection of cancer drug resistance and metastasis. Research has shown that it is possible to understand a cancer type and its drug resistance status from cellular permittivity and conductivity data. In fact, there is an increasing demand for analytical methods that can rapidly measure a cell’s electrical properties.
Electrorotation (ROT) offers one such route to capture cellular properties by inferring permittivity and conductivity from a cell’s movement in an electric field. This allows the characterization of the cell type and state by profiling its frequency-dependent rotational movement under a modulated electric field. However, there are limitations. The challenge is that the capture, measurement, and replacement of cells is quite cumbersome and lowers the throughput of ROT platforms, where throughput refers to the number of cells that a given technology can analyze at any given time.
Recently, researchers from Tokyo University of Science (TUS) developed a continuous flow ROT (cROT) to address conventional ROT’s drawbacks. The new platform leverages microfluidics to continuously measure cellular dynamics and simultaneously capture cells to collect measurements on one device. The group’s validated findings were recently published in Lab on a Chip on 23 October 2023.
“I discovered that cancer cells had vastly different responses to electric fields while they looked similar. This implied a certain degree of individuality, and the idea of discerning the differences using ROT intrigued me,” explains Dr. Masahiro Motosuke, a Professor in the Department of Mechanical Engineering at TUS and the project’s Principal Investigator. He further adds, “However, gathering accurate data using ROT requires the precise placement and removal of a single cell, and I wanted to make the process of analyzing many cells easier.”
The researchers fabricated the new device with redesigned interdigitating electrodes that induce cell rotation and a microchannel for cell passage. The electrode geometry increases the number of cells that can be analyzed and reduces the time required to replace a cell as measurements are collected. The electrical field applied within the microchannel enables analyzing rotational behavior from a continuous flow of cells. Together, these improvements increase the automated system’s throughput. The research team validated the system’s accuracy by obtaining cell membrane permittivity and cytoplasm conductivity measurements from HeLa cells, a human cell line commonly used in research.
“We significantly increased measurement throughput to 2,700 cells per hour with our cROT technique,” says Prof. Motosuke about the report’s most significant findings. “Furthermore, the device does not require precise cell manipulation and takes advantage of rapid image processing when processing the cells’ electrical data,” he adds further. Other advantages of the new system are its high degree of automation and ease of installation or removal.
The cROT device indeed demonstrates a remarkable enhancement in throughput when contrasted with traditional ROT platforms. While conventional ROT techniques typically process 10 to 20 cells per hour, the cROT system achieves an impressive throughput of 2700 cells per hour, which is more than 100 times higher. Furthermore, the cROT system significantly minimizes the time necessary for cell replacement.
Prof. Motosuke envisions a promising future for the cROT system that the team has developed. “With our cROT technique, we’ve unlocked the ability to delve into the subtle intricacies of single-cell dynamics, including aspects like cell physiology, the state of the cell membrane, and the concentration of intracellular ions,” he emphasizes. He anticipates that the swift and accurate analyses offered by this cutting-edge approach will be a catalyst for substantial advancements in the realms of cancer drug development, diagnosis, and novel cell-based therapies. This breakthrough technology opens doors for collaboration and adoption by prominent players in the oncology industry, potentially revolutionizing the way we combat cancer.

In mice and human cell cultures, MIT researchers showed that novel nanoparticles can deliver a potential therapy for inflammation in the brain, a prominent symptom in Alzheimer’s disease.
Some Covid-19 vaccines safely and effectively used lipid nanoparticles (LNPs) to deliver messenger RNA to cells. A new MIT study shows that different nanoparticles could be used for a potential Alzheimer’s disease (AD) therapy. In tests in multiple mouse models and with cultured human cells, a newly tailored LNP formulation effectively delivered small interfering RNA (siRNA) to the brain’s microglia immune cells to suppress expression of a protein linked to excessive inflammation in Alzheimer’s disease.
In a prior study the researchers showed that blocking the consequences of PU.1 protein activity helps to reduce Alzheimer’s disease-related neuroinflammation and pathology. The new results, reported in the journal Advanced Materials (impact factor 29.4 ) achieves a reduction in inflammation by directly tamping down expression of the Spi1 gene that encodes PU.1. More generally, the new study also demonstrates a new way to deliver RNA to microglia, which have been difficult to target so far.
Study co-senior author Li-Huei Tsai, Picower Professor of Neuroscience and Director of The Picower Institute for Learning and Memory and Aging Brain Initiative, said she hypothesized that LNPs might work as a way to bring siRNA into microglia because the cells, which clear waste in the brain, have a strong proclivity to uptake lipid molecules. She discussed this with Robert Langer, David Koch Institute Professor, who widely known for his seminal work on nanoparticle drug delivery, They decided to test the idea of reducing PU.1 expression with an LNP-delivered siRNA.
“I still remember the day when I asked to meet with Bob to discuss the idea of testing LNPs as a payload to target inflammatory microglia,” said Tsai, a faculty member in the Department of Brain and Cognitive Sciences. “I am very grateful to The JPB Foundation who supported this idea without any preliminary evidence.”
Langer Lab graduate student Jason Andresen and former Tsai Lab postdoc William Ralvenius led the work and are the study’s co-lead authors. Owen Fenton, a former Langer Lab postdoc who is now an assistant professor at the University of North Carolina’s Eshelman School of Pharmacy, is a co-corresponding author along with Tsai and Langer. Langer is a Professor in Chemical Engineering, Biological Engineering and the Koch Institute for Integrative Cancer Research.
Perfecting a particle
The simplest way to test whether siRNA could therapeutically suppress PU.1 expression would have been to make use of an already available delivery device, but one of the first discoveries in the study is that none of eight commercially available reagents could safely and effectively transfect cultured human microglia-like cells in the lab.

Instead the team had to optimize an LNP to do the job. LNPs have four main components and by changing the structures of two of them, and by varying the ratio of lipids to RNA, the researchers were able to come up with seven formulations to try. Importantly, their testing included trying their formulations on cultured microglia that they had induced into an inflammatory state. That state, after all, is the one in which the proposed treatment is needed.
Among the seven candidates, one the team named “MG-LNP” stood out for its especially high delivery efficiency and safety of a test RNA cargo.
What works in a dish sometimes doesn’t work in a living organism, so the team next tested their LNP formulations’ effectiveness and safety in mice. Testing two different methods of injection, into the body or into the cerebrospinal fluid (CSF), they found that injection into the CSF ensured much greater efficacy in targeting microglia without affecting cells in other organs. Among the seven formulations, MG-LNP again proved the most effective at transfecting microglia. Langer said he believes this could potentially open new ways of treating certain brain diseases with nanoparticles someday.
A targeted therapy
Once they knew MG-LNP could deliver a test cargo to microglia both in human cell cultures and mice, the scientists then tested whether using it to deliver a PU.1-suppressing siRNA could reduce inflammation in microglia. In the cell cultures, a relatively low dose achieved a 42 percent reduction of PU.1 expression (which is good because microglia need at least some PU.1 to live). Indeed MG-LNP transfection did not cause the cells any harm. It also significantly reduced the transcription of the genes that PU.1 expression increases in microglia, indicating that it can reduce multiple inflammatory markers.
In all these measures, and others, MG-LNP outperformed a commercially available reagent called RNAiMAX that the scientists tested in parallel.

“These findings support the use of MG-LNP-mediated anti-PU.1 siRNA delivery as a potential therapy for neuroinflammatory diseases,” the researchers wrote.
The final set of tests evaluated MG-LNP’s performance delivering the siRNA in two mouse models of inflammation in the brain. In one, mice were exposed to LPS, a molecule that simulates infection and stimulates a systemic inflammation response. In the other model, mice exhibit severe neurodegeneration and inflammation when an enzyme called CDK5 becomes hyperactivated by a protein called p25.
In both models, injection of MG-LNPs carrying the anti-PU.1 siRNA reduced expression of PU.1 and inflammatory markers, much like in the cultured human cells.
“MG-LNP delivery of anti-PU.1 siRNA can potentially be used as an anti-inflammatory therapeutic in mice with systemic inflammation an in the CK-p25 mouse model of AD-like neuroinflammation,” the scientists concluded, calling the results a “proof-of-principle.” More testing will be required before the idea could be tried in human patients.
In addition to Andresen, Ralvenius, Langer, Tsai and Owen, the paper’s other authors are Margaret Huston, Jay Penney and Julia Maeve Bonner.
In addition to the The JPB Foundation and The Picower Institute for Learning and Memory, the Robert and Renee Belfer Family, Eduardo Eurnekian, Lester A. Gimpelson, Jay L. and Carroll Miller, the Koch Institute, the Swiss National Science Foundation and the Alzheimer’s Association provided funding for the study.

A new study published in Proceedings of the National Academy of Sciences Nexus provides a better understanding of how the brain responds to injuries. Researchers at the George Washington University discovered that a protein called Snail plays a key role in coordinating the response of brain cells after an injury.
The study shows that after an injury to the central nervous system (CNS) a group of localized cells start to produce Snail, a transcription factor or protein that has been implicated in the repair process.The GW researchers show that changing how much Snail is produced can significantly affect whether the injury starts to heal efficiently or whether there is additional damage.
“Our findings reveal the intricate ways the brain responds to injuries,” said senior author Robert Miller, the Vivian Gill Distinguished Research Professor and Vice Dean of the GW School of Medicine and Health Sciences. “Snail appears to be a key player in coordinating these responses, opening up promising possibilities for treatments that can minimize damage and enhance recovery from neurological injuries.”
Key findings: This study identifies for the first time a special group of microglial-like cells that produce Snail. Microglial cells are found in the central nervous system. Lowering the amount of Snail produced after an injury results in inflammation and increased cell death. During this process the injury gets worse not better and there are fewer connections or synapses between brain cells. In contrast, when Snail levels are increased the outcome of brain injury improves-suggesting this protein can help limit the spread of injury-induced damage.The research raises questions about whether an experimental drug that affects Snail production could be used to limit the damage incurred after someone suffers a stroke or has been injured in an accident, Miller said. Additional studies must be done to show that increasing Snail production could curtail injury or even promote healing of the brain.
Miller and his team also plan to study the regulation of Snail in diseases like multiple sclerosis. Multiple sclerosis is a disease resulting in damage to myelin, the protective layer insulating nerve fibers in the brain. If drugs targeting Snail could be used to stop that damage, many of the future symptoms of this disease could be eased, he says.
But researchers have years of work to do before new drugs targeting Snail can be tested in clinical trials. The payoff ultimately might be drugs that can lead to accelerated healing for stroke damage, head wounds and even neurodegenerative diseases like dementia.
In addition to Miller and a team of researchers at the GW School of Medicine and Health Sciences, Cheryl Clarkson-Paredes, a senior research scientist at the GW Nanofabrication and Imaging Center, served as the lead author of the paper, “A unique cell population expressing the Epithelial-Mesenchymal Transition-transcription factor Snail moderates microglial and astrocyte injury responses,”
The research was supported by Biogen and the National Institutes of Health.

A Stanford Medicine-led, international study of hundreds of samples from patients with Hodgkin lymphoma has shown that levels of tumor DNA circulating in their blood can identify who is responding well to treatment and others who are likely to experience a disease recurrence — potentially letting some patients who are predicted to have favorable outcomes forgo lengthy treatment.
Surprisingly, the study also revealed that Hodgkin lymphoma, a cancer of the lymph nodes, can be divided into two groups, each with distinct genetic changes and slightly different prognoses. These changes hint at weaknesses in the cancer’s growth mechanisms that could be targeted by new, less toxic therapies.
The idea of establishing molecular profiles of tumors is not new. But unlike other cancers, Hodgkin lymphoma has resisted these types of analyses. That’s because Hodgkin lymphoma cells are relatively scarce — even within a large tumor.
“This approach offers our first significant look at the genetics of classical Hodgkin lymphoma,” said professor of medicine Ash Alizadeh, MD, PhD. “Compared with other cancers, finding Hodgkin lymphoma cancer cells or cancer DNA to study is like searching for a needle in a haystack. A patient can have a football-sized tumor in their chest, but only about 1% of the cells in the mass are cancer cells, with the remainder representing an inflammatory response to the tumor. This has made it very difficult to find the smoking guns that drive the disease.”
Alizadeh, who is the Moghadam Family Professor, and Maximilian Diehn, MD, PhD, professor of radiation oncology and the Jack, Lulu, and Sam Willson Professor, are the senior authors of the research, which will be published Dec. 11 in Nature. Former postdoctoral scholar Stefan Alig, MD; instructor of medicine Mohammad Shahrokh Esfahani, PhD; and graduate student Andrea Garofalo are the lead authors, as is graduate student Michael Yu Li at British Columbia Cancer.
About 8,500 people are diagnosed with Hodgkin lymphoma each year in the United States. The disease primarily affects people between the ages of 15 and 35, and people older than 55.
Stanford Medicine’s role
Just over 60 years ago, Stanford radiologist Henry Kaplan, MD pioneered the use of targeted radiation to treat Hodgkin lymphoma. The new therapy, delivered by a high-energy linear accelerator Kaplan developed in the 1950s for medical use, was the first step in a Stanford-driven effort to turn the once fatal cancer of the lymph nodes into one that is now highly curable. Soon thereafter, Kaplan was joined by medical oncologist Saul Rosenberg, MD, and the two worked out ways to combine radiation therapy with chemotherapy regimens, including one known simply as the Stanford 5 (named because it was the fifth in a series of gradually less toxic treatments).

During the subsequent decades, however, the genetic changes that cause the cancer have remained mysterious. That’s because, unlike many other cancers, Hodgkin lymphoma tumors are made up primarily of immune cells that have infiltrated the cancer, making it difficult to isolate the diseased cells for study. Today, patients are treated with chemotherapy, radiation or a combination of both; about 89% of patients survive five years or more after their initial diagnosis.
Alizadeh, Diehn and their colleagues used an optimized DNA sequencing technique called PhasED-Seq, or phased variant enrichment and detection sequencing, they developed at Stanford Medicine in 2021 to home in on vanishingly rare bits of DNA in a patient’s bloodstream to identify genetic changes that drive the growth of Hodgkin lymphoma.
PhasED-Seq builds upon a technique called CAPP-Seq, or cancer personalized profiling by deep sequencing,developed in 2014 by Alizadeh and Diehn to assess lung cancer levels and response to treatment. But PhasED-Seq is much more sensitive.
“CAPP-Seq could detect as few as one cancer DNA sequence in 10,000 non-cancer DNA sequences,” Diehn said. “But PhasED-Seq can detect less than one cancer DNA sequence in 1 million non-cancer DNA sequences.”
Their goal was to learn more about what drives the cancer and how to make successful treatments even easier for patients.
“We typically can cure most patients with one line of therapy,” Alizadeh said. “But we are always trying to figure out less toxic chemotherapeutic agents that are gentler to the bone marrow, lungs and other organs, and ways to more precisely target radiation therapy. And a small minority of patients experience a recurrence that can be challenging to treat successfully.”
The researchers used CAPP-Seq and PhasED-Seq to analyze blood samples from 366 people treated for Hodgkin lymphoma at three medical centers including Stanford Medicine. The technique was remarkably sensitive.

“Surprisingly, we detected more cancer DNA in the blood than in the cancer tissue itself,” Alizadeh said. “That seemed hard to believe until we had analyzed enough samples to show that it was reproducible.”
Two paths
The researchers used machine learning techniques to categorize the different types of genetic changes present in the cancer cells. They found that patients could be separated into two groups: one that predominantly has mutations in several cancer-associated genes implicated in cellular survival, growth and inflammation, and another with a type of genetic change called copy number alterations that affects larger swathes of the genome, subbing in or excising regions of DNA that influence cell growth and cancer.
“We adapted a method from natural language processing to find these two Hodgkin subtypes, and then used a variety of methods to identify key biological and clinical features and to confirm that the subtypes are also seen in other groups of patients,” Esfahani said.
The first group, which makes up about one-half to two-thirds of patients, occurs primarily in younger patients and has a comparatively more favorable outcome. About 85-90% of these people survive for three years without disease recurrence. The second, which makes up about one-half to one-third of the total, occurs in both younger and older patients and has a lessfavorable, although still good outcome. About 75% of these people live for at least three years without recurrence.
Critically, a subset of both groups contained a unique mutation in a gene for the receptor for cellular signaling proteins called interleukin 4 and interleukin 13.
“We discovered a new class of mutations in the interleukin 4 receptor gene that enhance a key pathway characteristic to Hodgkin lymphoma,” Alig said. “These mutations may be indicative of unique vulnerabilities of the tumor that can be exploited therapeutically.”
The researchers also showed that patients who had no detectable circulating tumor DNA in their blood shortly after starting treatment were much less likely to have disease recurrence than those who had even small amounts of residual circulating cancer DNA at the same time point — a distinction researchers had hoped to see, but were unsure about being able to detect even with PhasED-Seq.
“I was surprised that we could predict which patients would recur,” Diehn said. “Even with our ultrasensitive assay there was a significant chance that the signal from the cancer DNA could become undetectable after treatment, even in patients who eventually recurred. But this didn’t happen.”
The researchers seeking to understand more about the biology of Hodgkin lymphoma have one key goal: the improvement of care for patients.
“The number of people who experience recurrence is small, but, like Henry Kaplan and Saul Rosenberg, we want to save every one of them,” Diehn said. “They would have been amazed and gratified by these findings, which build upon their important work from decades ago. We look forward to an era in which we can cure every patient with no toxicity.”
Researchers from British Columbia Cancer, University Hospital François Mitterand, St. Jude Children’s Research Hospital, the Oncology Institute of Southern Switzerland, KU Leuven, the University of Strasbourg, Emory University, the Fred Hutchinson Cancer Research Center, the Hospices Civils de Lyon, and the Université Catholique de Louvain contributed to the work.
This work was supported by the National Institutes of Health (grants R01CA257655, R01CA233975 and R03CA21765), the Department of Defense, the Virginia and D.K. Ludwig Fund for Cancer Research, a Hanna and Michael Murphy family gift, the Stanford Cancer Institute, the Damon Runyon Cancer Research Foundation, an American Society of Hematology Scholar Award, the V Foundation for Cancer Research, the Emerson Collective Cancer Research Fund, a Stinehart/Reed Award, the CRK Faculty Scholar Fund, the Lung Cancer Research Foundation and the SDW/DT, the Shanahan Family Foundations, the Terry Fox Research Institute, the Canadian Institutes of Health Research, an Elizabeth C. Watters Research Fellowship, and the American Syrian Lebanese Associated Charities.
Diehn, Alizadeh, Alig and Esfahani have filed patents related to cancer biomarkers. Diehn has multiple issued and pending patents licensed to Foresight Diagnostics and Roche. He holds interests in CiberMed, Inc.; Foresight Diagnostics; and Gritstone Bio. Alizadeh has multiple issued and pending patents licensed to Foresight Diagnostics, CiberMed Inc. and Idiotype Vaccines. He holds interests in CiberMed, Inc.; Foresight Diagnostics; FortySeven Inc.; and CARGO Therapeutics.

The Organoid Group (Hubrecht Institute) and the Rare Cancers Genomics Team (IARC/WHO) found a way to grow samples of different types of neuroendocrine tumors (NETs) in the lab. While generating their new model, the researchers discovered that some pulmonary NETs need the protein EGF to be able to grow. These types of tumors may therefore be treatable using inhibitors of the EGF receptor. The results were published in Cancer Cell on 11 December 2023.
Neuroendocrine tumors
Neuroendocrine tumors (NETs) are relatively rare tumors that can be slow-growing. However, some NETs can be aggressive and hard to treat. It is not yet possible to predict which tumors will become aggressive. There are very few models to study NETs in the lab, which limits research into this type of tumor.
New disease model
Researchers from the Organoid Group (Hubrecht Institute) and the Rare Cancers Genomics Team (IARC/WHO) therefore set out to develop new models to study NETs. They derived cells from patients with NETs and were able to culture them into 3D structures called organoids. These organoids mimic the behavior of actual NETs and can therefore be used to study this type of tumor in the lab. The new model is the first organoid model of the disease.
Growth factor
While generating the organoids, the researchers found that some pulmonary NETs need a protein called the Epidermal Growth Factor (EGF) to grow. “If we inhibit the receptor for EGF, some organoids die. Apparently, these organoids are dependent on EGF for their survival,” says Talya Dayton, co-first author on the paper published in Cancer Cell. “We need further research to confirm our findings, but this may indicate that patients with EGF-dependent NETs could be treated with inhibitors of the EGF receptor.” Inhibitors of the EGF receptor are already a course of treatment for other types of tumors.

Aggressive tumors
Tumors are usually thought to be independent of growth factors. That some NETs turn out to be dependent on the growth factor EGF is therefore surprising. “We think that their EGF-dependence might explain, in part, why some of these tumors grow slowly. We also think this might mean that one of the ways in which NETs can become aggressive is by becoming growth-factor independent. If they no longer need the growth factor, their growth may accelerate” Dayton explains.
Potential new therapy
The newly developed model for NETs provides a new way to study the disease in the lab. Dayton: “This allows us and other scientists to understand the biology of these tumors so we can hopefully find effective therapies.” Although further research is needed, the model already points to a new route of treatment for patients with pulmonary NETs.