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Bone-Enhancing Peptide Shows Promise as Therapeutic for Osteoporosis and MSK Disorders

Studies by University of Birmingham researchers suggest that a naturally occurring peptide known as PEPITEM (Peptide Inhibitor of Trans-Endothelial Migration), could represent a promising potential therapeutic for osteoporosis and other disorders that feature bone loss, and offer distinct advantages compared with existing drugs.

PEPITEM was first identified in 2015 by University of Birmingham researchers. The team’s latest study has shown for the first time that PEPITEM could be used as a novel and early clinical intervention to reverse the impact of age-related musculoskeletal diseases. Their reported study demonstrated that PEPITEM enhances bone mineralization, formation and strength, and reverses bone loss in animal models of disease.

Helen McGettrick, PhD, associate professor in inflammation and vascular biology said, “While the most commonly used drugs, bisphosphonates, work by blocking the action of osteoclasts, PEPITEM acts by swinging the balance in favor of bone formation, without impacting the ability of osteoclasts to resorb regions of damaged or weak bone tissue via normal bone remodeling.”

McGettrick is senior, and corresponding author of the team’s published paper in Cell Reports Medicine. In their paper, titled “Therapeutic avenues in bone repair: Harnessing an anabolic osteopeptide, PEPITEM, to boost bone growth and prevent bone loss,” the investigators concluded, “… PEPITEM offers an alternative therapeutic option in the management of diseases with excessive bone loss, promoting an endogenous anabolic pathway to induce bone remodeling and redress the imbalance in bone turnover.”

Bone is constantly formed, reformed, and remodelled throughout life, and up to 10 percent of human bone is replaced annually through a complex interplay between two cell types— osteoblasts, which form bone, and osteoclasts, which breakdown bone. “Bone is a highly active organ, undergoing continuous osteoblast-induced bone formation and osteoclast-mediated bone resorption throughout life,” the authors explained. “The process of bone remodeling is orchestrated by cross-talk among osteoblasts, osteoclasts, and osteocytes acting in concert to maintain structural integrity, repair damage, and respond to changes in activity and load.”

Disturbances to this tightly orchestrated process are responsible for features of musculoskeletal (MSK) diseases such as osteoporosis, rheumatoid arthritis, and cancer-bone metastases, which show excessive bone breakdown, or ankylosing spondylitis, which is characterized by abnormal bone growth.

Osteoporosis is the most common bone disease globally, affecting over 54 million individuals in the U.S., and accounting for three million broken bones at a cost of $26 billion per annum, the team stated. “There are no cures for bone damage.” The most commonly used osteoporosis therapies (bisphosphonates) target osteoclasts to prevent further bone loss. Although there are new ‘anabolic’ agents that can promote new bone formation, these have limitations in their clinical use, with teriparatide (parathyroid hormone; PTH) only being effective for 24 months and romosozumab (anti-sclerostin antibody) being associated with cardiovascular events.

There is a clear case for developing new therapies to stimulate bone repair in age-related musculoskeletal diseases, including osteoporosis, the researchers pointed out. “… there is an urgent need to develop a new suite of therapies that lead to bone repair and regeneration in patients with MSK diseases to restore tissue homeostasis and functional integrity.” For their reported work the team—headed by McGettrick and Amy Naylor, PhD, Jonathan Lewis, PhD, and Kathryn Frost, from the Institute of Inflammation and Ageing at the University of Birmingham, and James Edwards, PhD, from Nuffield Department of Orthopedics, Rheumatology and Musculoskeletal Sciences at the University of Oxford—set out to investigate the potential therapeutic impact of PEPITEM in these disease states.

PEPITEM is a naturally occurring peptide that is produced in the body and circulates at low levels. Through their studies in mice, the team demonstrated that PEPITEM regulates bone remodeling, and that increasing the amount present in the body stimulated bone mineralization in “young bones” that are not in a diseased or pre-osteoporotic state. “PEPITEM therapy significantly increased bone volume (BV/TV), trabecular number, and thickness in both the tibia and vertebrae of adult mice, indicating that PEPITEM promotes bone formation.” This, they found, translated to an increase in bone strength and density similar to that promoted by current standard of care drugs (bisphosphonates and PTH). “… the effect size for PEPITEM on BV/TV at two weeks is comparable to that seen following treatment with the bisphosphonate zoledronic acid for three weeks  or PTH for up to four weeks.Thus indicating PEPITEM is as efficient at inducing bone formation compared with current standard of care,” they noted.

A key test for a potential new therapeutic is its ability to target the natural repair process that is compromised by age, or inflammatory disease. Through their work the researchers showed that PEPITEM administration limited bone loss and improved bone density in ovariectomized animal models of the menopause, which is a common trigger for osteoporotic bone loss in humans. “Crucially, PEPITEM therapy halted any further bone loss following ovariectomy,” they wrote, and was effective at inducing bone formation by osteoblasts isolated from aged donors with osteoarthritis.” Their studies also showed similar findings in models of inflammatory bone disease (arthritis), where PEPITEM significantly reduced bone damage and erosion. “Similar findings were seen in an inflammatory model of bone erosion, where PEPITEM treatment significantly reduced bone damage in arthritic mice when compared with vehicle-treated animals.”

The findings in mice were supported by the results of work using human bone tissue, harvested from older patients during joint surgery. These studies showed that cells from older individuals respond to PEPITEM, significantly increasing the maturation of osteoblasts, and their ability to produce and mineralise bone tissues.

The team’s cell and tissue culture work further showed PEPITEM has a direct effect on osteoblasts to promote bone formation, by increasing the activity of osteoblasts rather than their number. Further experiments identified the NCAM-1 receptor as the specific receptor for PEPITEM on osteoblasts, and strongly suggested the NCAM-1- β-catenin signalling pathway is responsible for the upregulation of osteoblast activity. “PEPITEM acts directly on osteoblasts through NCAM-1 signaling to promote their maturation and formation of new bone, leading to enhanced trabecular bone growth and strength,” they wrote. This receptor, and the pathway, are distinct from PEPITEM receptors that have been previously described in other tissues.

The researchers also investigated PEPITEM’s effect on osteoclasts and bone resorption.  Here, mouse studies showed that PEPITEM significantly reduces the number of osteoclasts, leading to reduced bone mineral resorption. “Analysis of bone sections from mice treated with PEPITEM revealed a significant reduction in osteoclast numbers when compared with the treatment controls …” The researchers subsequently demonstrated that the reduction in osteoclast activity is the result of a soluble substance, osteoprotegerin (OPG) released locally in bone tissues by osteoblasts ‘activated’ by PEPITEM. The collective data, they stated, “…  indicate that in response to PEPITEM signaling through NCAM-1, osteoblasts release OPG, which in turn negatively regulate osteoclast numbers, leading to an overall reduction in bone resorption and increase in bone density.”

The study results, the team stated, “… highlight that PEPITEM could be used as an alternative and early clinical intervention to reverse the impact of age-related MSK diseases … As an endogenous osteogenic peptide with the capacity to regulate osteoblast-osteoclast coupling in health and disease, PEPITEM offers the real possibility for maintenance or restoration of bone homeostasis over the long-term to prevent osteoporosis and fragility fractures. For this to be realized, prolonged treatment protocols are now required in a variety of bone disease models to ascertain the quality of the bone formed in response to PEPITEM therapy.”

Cannibalism Genes in Human Genome Evolved to Serve New Cellular Functions

It’s a cell-eat-cell world out there—and not just for single-celled organisms. Indeed, even people have something of the cannibal in their cells, even cells that have nothing to do with malignancy or immune function.

What we know about cell-eat-cell phenomena—or, more generally, cell-in-cell phenomena—has been reviewed by scientists based at Arizona State University. They performed a systematic screening of 508 articles, from which they chose 115 relevant articles in a search for cell-in-cell events across the tree of life, the age of cell-in-cell-related genes, and whether cell-in-cell events are associated with normal multicellular development or cancer.

The scientists, led by Carlo C. Maley, PhD, director of the Arizona Cancer and Evolution Center, presented their findings in Scientific Reports, in a paper titled, “Cell-in-cell phenomena across the tree of life.”

“Cell-in-cell events are found across the tree of life, from some unicellular to many multicellular organisms, including non-neoplastic and neoplastic tissue,” the article’s authors wrote. “Additionally, out of the 38 cell-in-cell-related genes found in the literature, 14 genes were over 2.2 billion years old, that is, older than the common ancestor of some facultatively multicellular taxa. All of this suggests that cell-in-cell events may have originated before the origins of obligate multicellularity.”

The widespread occurrence of interactions in which cells become internalized by other cells suggests that these events are not inherently “selfish” or “cancerous.” Rather, it appears that cell-in-cell phenomena may play crucial roles in normal development, homeostasis, and stress response across a wide range of organisms.

“[Our] results show that cell-in-cell events exist in obligate multicellular organisms, but are not a defining feature of them,” the scientists emphasized. “The idea of eradicating cell-in-cell events from obligate multicellular organisms as a way of treating cancer, without considering that cell-in-cell events are also part of normal development, should be abandoned.”

By demonstrating that occurrences span a wide array of life forms and are deeply rooted in our genetic makeup, the research invites us to reconsider fundamental concepts of cellular cooperation, competition, and the intricate nature of multicellularity. The study opens new avenues for research in evolutionary biology, oncology, and regenerative medicine.

“We first got into this work because we learned that cells don’t just compete for resources—they actively kill and eat each other,” Maley said. “That’s a fascinating aspect of the ecology of cancer cells. But further exploration revealed that these phenomena happen in normal cells, and sometimes neither cell dies, resulting in an entirely new type of hybrid cell.”

The study describes 16 different taxonomic groups in which cell-in-cell behavior is found to occur. The cell-in-cell events were classified into six distinct categories based on the degree of relatedness between the host and prey cells, as well as the outcome of the interaction (whether one or both cells survived).

A spectrum of cell-in-cell behaviors is highlighted in the study, ranging from completely selfish acts, where one cell kills and consumes another, to more cooperative interactions, where both cells remain alive. For example, the researchers found evidence of “heterospecific killing,” where a cell engulfs and kills a cell of a different species, across a wide range of unicellular, facultatively multicellular, and obligate multicellular organisms. In contrast, “conspecific killing,” where a cell consumes another cell of the same species, was less common, observed in only three of the seven major taxonomic groups examined.

In addition to cataloging the diverse cell-in-cell behaviors, the researchers also investigated the evolutionary origins of the genes involved in these processes. Surprisingly, they found that many of the key cell-in-cell genes emerged long before the evolution of obligate multicellularity.

“When we look at genes associated with known cell-in-cell mechanisms in species that diverged from the human lineage a very long time ago, it turns out that the human orthologs (genes that evolved from a common ancestral gene) are typically associated with normal functions of multicellularity, like immune surveillance,” said study co-author Luis Cisneros, PhD, formerly a research assistant professor at Arizona State University and currently a research associate at Mayo Clinic.

The ancient cell-in-cell genes identified in the study are involved in a variety of cellular processes, including cell–cell adhesion, phagocytosis (engulfment), intracellular killing of pathogens, and regulation of energy metabolism. This diversity of functions indicates that cell-in-cell events likely served important roles even in single-celled and simple multicellular organisms well before the emergence of complex multicellular life.

Bud-wiser: DOD Backs A-Alpha Bio for Biothreat Preparation

As a graduate student in the lab of protein design expert David Baker, PhD, at the University of Washington, David Younger, PhD, noticed a major bottleneck to designing massive amounts of proteins in silico: how does anyone test so many proteins in silico? 

“You would have someone in the lab designing tens or hundreds of thousands of proteins; they might only be able to test 10 of them, or maybe 100 if the project is further along,” Younger told GEN Edge. Even then, you’re only measuring binding against one thing. You’re not measuring specificity or cross-reactivity and not determining the epitope of exactly where that protein is binding.” 

Younger began to ponder how to use synthetic biology and machine learning to engineer a cellular system to generate and analyze large multiplex data sets around how proteins bind to each other.

“With any kind of biological technology, you go in hoping for a paper, and anything else is kind of icing on the cake,” said Younger. “In this case, we were happily surprised, three or four years later, that this platform is really working. This has implications for helping computational protein designers test designs with higher throughput, and there are some real, near-term pharma applications.”  

In 2017, Younger spun out his research into a company called A-Alpha Bio, together with Randolph Lopez, PhD, to use their platform, called AlphaSeq, to develop an in-house therapeutic pipeline and form partnerships with pharmaceutical companies to inform the discovery and development of novel therapeutics. But Younger’s interests aren’t just limited to pharma—he’s also been interested in mitigating potential future biothreats. 

In 2022, A-Alpha Bio received funding from the Department of Defense’s (DOD) Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense’s (JPEO-CBRND’s) Generative Unconstrained Intelligent Drug Engineering (GUIDE) program to preemptively generate data and train computational models that enable rapid medical countermeasures against potential future biothreats.  

The DOD’s initial investment up through 2023 had been $3.4 million, and now funding has expanded as A-Alpha Bio has been awarded another $14.5 million to accelerate antibody discovery and optimization for likely biothreats in partnership with the Lawrence Livermore National Laboratory (LLNL), a federally funded research and development center. With this extra help, A-Alpha Bio will be able to make large datasets of antibody-antigen binding and train and test predictive computer models for unknown pathogen families of concern. 

“We’ve measured 10 million interactions in this collaboration and have focused so far on three pathogenic families,” said Younger. “This extension of funding will allow us to really broaden both our potential impact on pandemic preparedness and our interest in training more generalizable models. Having an interaction space that covers more pathogen diversity is exactly what we’re interested in doing.”  

What’s love got to do with it? 

At the heart of Younger’s synthetic biology approach to measuring very large numbers of protein interactions with quantitative accuracy are two simple concepts: yeast phage display and mating. 

Essentially, Younger built two distinct yeast surface display libraries—one with thousands of unique antibodies and the other with variants and homologs of antigens. The two libraries, which each have a different yeast sex (i.e., A and alpha), are swirled together in a liquid culture where the yeast collide. Cells will stick and then fuse if there is a sufficiently strong interaction.  

Once the culture has grown, the fused yeast can be taken out and sequenced to be counted. This shows the protein-protein interactions and how strong each pairwise interaction is by measuring how often the yeast fuses. Younger claims that, to gather enough data for machine learning to analyze to optimize crucial binding properties, the process frequently necessitates several cycles of generating libraries and screening. 

The yeast libraries can be custom-generated for different applications, such as cancer, which is the therapeutic focus for A-Alpha Bio. For an oncology program, the antigen library can consist of a huge catalog of variants of a particular protein of interest. However, because the data is so quantitative and enables multivariable optimization of critical binding properties, AlphaSeq is attractive for screening potential synthetic biothreats. 

“We want to take every possible viral family that is a possible future pandemic, either naturally derived or potentially weaponized, and we want to essentially generate data as if there were a pandemic in that family,” said Younger. “We want to understand how antibodies bind to diverse antigens within that antigenic family. We also want to train predictive machine learning models so that we can more easily respond if a new variant within that viral family crops up. It’s hard to generate many antibodies against many antigens with any other platform.” 

Data, the digital diamonds 

One aspect of the partnership with the DOD that Younger is particularly excited about is having full usage rights for the expansive database they are building. It is approaching one billion protein interaction measurements, which Younger says is several orders of magnitude larger than anything that exists, such as the Biological General Repository for Interaction Datasets (BioGRID) database, which has several million entries. 

“The most exciting companies combine exciting proprietary sources of data that give new insights with sophisticated computation,” said Younger. Our work with groups like LLNL, DOD, and JPEO-CBRND allows us to continue to invest in this massive data generation campaign, which we can use to leverage for all other applications that we’re interested in as a company.” 

Even though the biosecurity work doesn’t overlap with the focus of A-Alpha Bio’s therapeutic pipeline, Younger said that these data can still be used to train their own machine learning models and be applied to their drug development campaigns in areas like oncology that are still in their early discovery phases. 

“We’re looking at an experimental tool that allows us to generate these really comprehensive, highly quantitative, vast data sets that are just impossible to generate using any conventional technologies and then layering machine learning on top of that actually to generate the practical insights and have that impact that we desire to have,” said Younger

“It’s a really exciting time for the field, and we’re excited to be a part of it.” 

Severe COVID-19 Lung Disease Linked to Ferroptosis

Over the past few years, researchers have uncovered countless details about how SARS-CoV-2 causes COVID-19. However, the mechanisms underlying how SARS-CoV-2 infection causes severe pulmonary manifestations remain poorly understood. This, in turn, limits treatment options.

In some severe cases of COVID-19, the lungs undergo extreme damage, resulting in a range of life-threatening conditions like pneumonia, inflammation, and acute respiratory distress syndrome. The root cause of those wide-ranging reactions in the lungs has remained unclear. However, hyperferritinemia and disrupted lung iron homeostasis in COVID-19 patients have pointed to ferroptosis—an iron-dependent cell death.

Now, a new study finds that ferroptosis is the major cell death mechanism that underlies COVID-19 lung disease. In a new paper, the authors noted that “immunostaining and lipidomic analysis in COVID-19 lung autopsies reveal increases in ferroptosis markers, including transferrin receptor 1 and malondialdehyde accumulation in fatal cases.” The finding indicates that deliberately halting ferroptosis with therapeutic drug candidates could improve COVID-19 outcomes.

This work is published in Nature Communications in the paper, “Fatal COVID-19 pulmonary disease involves ferroptosis.

“This finding adds crucial insight to our understanding of how COVID-19 affects the body that will significantly improve our ability to fight life-threatening cases of the disease,” said Brent Stockwell, PhD, chair of the department of biological sciences and a professor at Columbia University in the departments of biological science and chemistry.

COVID-19 lungs displayed dysregulation of lipids involved in metabolism and ferroptosis. The researchers found increased ferritin light chain associated with severe COVID-19 lung pathology. More specifically, the authors noted that “iron overload promotes ferroptosis in both primary cells and cancerous lung epithelial cells. In addition, ferroptosis markers strongly correlate with lung injury severity in a COVID-19 lung disease model using male Syrian hamsters.”

Ferroptosis was first reported by Stockwell in 2012. Ferroptosis differs from the most common kind of cell death, which occurs both in disease contexts and in normal processes like aging and involves cells chopping up the molecules in their interior.

Though ferroptosis can be destructive, recent studies indicate that intentionally inducing ferroptosis could counteract diseases like cancer where rampant cell growth is dangerously occurring. The ability to inhibit ferroptosis, on the other hand, could offer doctors new ways of combating cell death that should not be occurring, as in the case of COVID-19 lung disease.

“We’re hopeful that these important new findings could improve our ability to confront this pernicious disease, which, in too many cases, still diminishes health outcomes and results in death,” Stockwell said.

Autoimmune Disease Prediction Boosted by AI

Researchers at Penn State College of Medicine have developed a new artificial intelligence algorithm that may lead to improved predictions and novel therapies for autoimmune diseases. The algorithm dives into the genetic code underlying the conditions to more accurately model how genes associated with specific autoimmune diseases are expressed and regulated and to identify additional genes of risk.

Their findings are published in Nature Communications in an article titled, “Integrating single cell expression quantitative trait loci summary statistics to understand complex trait risk genes.”

“Transcriptome-wide association study (TWAS) is a popular approach to dissect the functional consequence of disease associated non-coding variants,” the researchers wrote. “Most existing TWAS use bulk tissues and may not have the resolution to reveal cell-type specific target genes. Single-cell expression quantitative trait loci (sc-eQTL) datasets are emerging. The largest bulk- and sc-eQTL datasets are most conveniently available as summary statistics, but have not been broadly utilized in TWAS. Here, we present a new method EXPRESSO (EXpression PREdiction with Summary Statistics Only), to analyze sc-eQTL summary statistics, which also integrates 3D genomic data and epigenomic annotation to prioritize causal variants.”

The researchers report that their algorithm outperforms existing methodologies and identified 26% more novel gene and trait associations.

“We all carry some DNA mutations, and we need to figure out how any one of these mutations may influence gene expression linked to disease so we can predict disease risk early. This is especially important for autoimmune disease,” explained Dajiang Liu, PhD, distinguished professor, vice chair for research, and director of artificial intelligence and biomedical informatics at the Penn State College of Medicine and co-senior author of the study. “If an AI algorithm can more accurately predict disease risk, it means we can carry out interventions earlier.”

EXPRESSO applies a more advanced artificial intelligence algorithm and analyzes data from single-cell expression quantitative trait loci, a type of data that links genetic variants to the genes they regulate. It also integrates 3D genomic data and epigenetics—which measures how genes may be modified by environment to influence disease—into its modeling. The team applied EXPRESSO to GWAS datasets for 14 autoimmune diseases, including lupus, Crohn’s disease, ulcerative colitis, and rheumatoid arthritis.

“With this new method, we were able to identify many more risk genes for autoimmune disease that actually have cell-type specific effects, meaning that they only have effects in a particular cell type and not others,” said Bibo Jiang, PhD, assistant professor at the Penn State College of Medicine and senior author of the study.

The team then used this information to identify potential therapeutics for autoimmune disease.

“Most treatments are designed to mitigate symptoms, not cure the disease. It’s a dilemma knowing that autoimmune disease needs long-term treatment, but the existing treatments often have such bad side effects that they can’t be used for long. Yet, genomics and AI offer a promising route to develop novel therapeutics,” said Laura Carrel, PhD, professor of biochemistry and molecular biology at the Penn State College of Medicine and co-senior author of the study.

The team’s work pointed to drug compounds that could reverse gene expression in cell types associated with an autoimmune disease, such as vitamin K for ulcerative colitis and metformin, which is typically prescribed for type 2 diabetes. These drugs, already approved by the FDA as safe and effective for treating other diseases, could potentially be repurposed.

The research team is working with collaborators to validate their findings in a laboratory setting and, ultimately, in clinical trials.

CRISPR Screen Broadens and Quickens Study of Gene Function across Cell Types

A new CRISPR screen method developed at Scripps Research has the potential to improve studies into the genetic underpinnings of human diseases and disorders. The method was outlined in Cell, in a paper titled, “Massively parallel in vivo Perturb-seq reveals cell-type-specific transcriptional networks in cortical development.”

The paper’s authors, led by Xin Jin, PhD, a neuroscientist at Scripps Research, summarized the limitations of existing CRISPR screens: 1) They have difficulty accomplishing the scalable labeling and perturbation of sufficient numbers of cells in vivo. 2) They have difficulty deconvoluting each cell’s perturbation identity in the sparse single-cell omics data. 3) They rely on lentiviral vectors, which are known to have limited in vivo penetration and thermostability, hampering systemic screens in hard-to-reach tissues such as the central and peripheral nervous systems.

To address these issues, Jin and colleagues developed an AAV-based, massively parallel in vivo Perturb-seq platform to target broad tissues and cell types with gene-expression-based characterization at single-cell resolution. To demonstrate the platform, the scientists used it to study the development of embryonic brains in mice.

“Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons,” the article’s authors wrote. “Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq.”

The view of the brain with the perturbation expression. [Scripps Research]
Jin explained that the new method could help researchers investigate diseases in more detail: “We know that certain genetic variations in our genome can make us vulnerable or resilient toward different diseases, but which specific cell types are behind a disease? Which brain regions are susceptible to the genomic mutations in those cells? These are the kinds of questions we’re trying to answer.

“With this new technology, we want to build a more dynamic picture across brain region, across cell type, across the timing of disease development, and really start understanding how the disease happened—and how to design interventions.”

Thanks to over a decade’s efforts in human genetics, scientists have had access to long lists of genetic changes that contribute to a range of human illnesses, but knowing how a gene causes a disease is very different than knowing how to treat the illness itself. Every risk gene may impact one or several different cell types. Comprehending how those cell types—and even individual cells—impact a gene and affect disease progression is key to understanding how to ultimately treat that disease.

This is why Jin, along with the study’s first author, Xinhe Zheng, a PhD candidate and the Frank J. Dixon Graduate Fellow at Scripps Research, co-invented the new technique, named in vivo Perturb-seq. This method leverages CRISPR-Cas9 technology and a readout, single-cell transcriptomic analysis, to measure its impact on a cell: one cell at a time. Using CRISPR-Cas9, scientists can make precise changes to the genome during brain development, and then closely study how those changes affect individual cells using single-cell transcriptomic analysis—for tens of thousands of cells in parallel.

“Our new system can measure individual cells’ response after genetic perturbations, meaning that we can paint a picture of whether certain cell types are more susceptible than others and react differently when a particular mutation happens,” Jin said.

Previously, the method for introducing the genetic perturbations into the brain tissue was very slow, often taking days or even weeks, which created suboptimal conditions for studying gene functions related to neurodevelopment. But Jin’s new screening method allows for rapid expression of perturbation agents in living cells within 48 hours—meaning scientists can quickly see how specific genes function in different types of cells in a very short amount of time.

The method also enables a level of scalability that was previously impossible—the research team was able to profile more than 30,000 cells in just one experiment, 10–20 times accelerated from the traditional approaches. In many of the brain regions they examined, such as the cerebellum, they were able to collect tens of thousands of cells that previous labeling methods could not reach.

In a pilot study using this new technology, Jin and her team’s interest was piqued when they saw a genetic perturbation elicit different effects when perturbed in different cell types. This is important because those impacted cell types are the sites of action for particular diseases or genetic variants. “Despite their smaller population representations, some low-abundant cell types may have a stronger impact than others by the genetic perturbation, and when we systematically look at other cell types across multiple genes, we see patterns. That’s why single-cell resolution—being able to study every cell and how each one behaves—can offer us a systematic view,” Jin said.

With her new technology in hand, Jin plans to apply it to better understand neuropsychiatric conditions and how certain cell types correspond with various brain regions. Moving forward, Jin said she’s excited to see this type of technology applied to additional cell types in other organs in the body to better understand a wide range of diseases in terms of tissue, development, and aging.

Tumor-Targeting, Drug-Loaded Extracellular Vesicles Improve Survival in Cancer-Bearing Mice

Researchers at the Karolinska Institute have succeeded in delivering targeted cancer treatment via extracellular vesicles (EVs), the small membrane bubbles that our cells use to communicate. Using molecular engineering tools, the team developed EVs that can bind the fragment crystallizable (Fc) portion of antibodies, so that the variable regions are displayed for antigen recognition. These Fc-binding EVs (Fc-EVs) can be decorated with different types of antibody, to potentially target tissues of interest. In their newly reported study in cancer-bearing mice, the team demonstrated reduced tumor growth and improved survival among animals treated using engineered Fc-EVs studded with tumor-targeting PD-L1 antibodies and loaded with a chemotherapy cargo.

“By attaching different antibodies to extracellular vesicles, we can target them to virtually any tissue and we can load them with other types of drugs as well,” said Oscar Wiklander, PhD, at the department of laboratory medicine, Karolinska Institute. “Therefore, the treatment has the potential to be used against other diseases and cancer types.” Wiklander is co-first author of the team’s published paper in Nature Biomedical Engineering, which is titled “Antibody-displaying extracellular vesicles for targeted cancer therapy.”

When our cells communicate, they send out small membrane bubbles known as extracellular vesicles, which contain various signaling molecules. “EVs contain lipids, proteins, and nucleic acid species from the source cell, and have the unique ability to convey these macromolecules via an advanced system of intercellular communication,” the authors explained. Interest in these nanovesicles, sometimes referred to as the body’s “message in a bottle,” has increased in recent years as they represent “promising nanocarriers for drug delivery,” the team continued. “Compared to other vehicles, such as liposomes and polymer-based synthetic nanoparticles, EVs are associated with high biocompatibility, minimal toxicity and enhanced drug potency, with improved pharmacokinetic profiles including improved tumor penetrance, and retention in tumor cells,” the team indicated. “Importantly, EVs benefit from the ability to cross biological barriers to reach distant organs and can be engineered to display targeting moieties and loaded with a wide variety of therapeutic cargo molecules.”

In recent years, EVs have gained increasing attention and there are currently numerous clinical trials being undertaken to evaluate the therapeutic potential of EVs, the investigators further noted. For their reported study the team set out to investigate whether Fc-EVs could be used as a delivery vehicle for targeted delivery of the chemotherapy drug doxorubicin (Dox) to tumor-bearing mice. They created a targeted cancer treatment by loading EVs with the chemotherapeutic drug and attaching tumor-targeting antibodies (Abs) to their surfaces, to create an Fc-Ev+Dox+PD-L1-Ab construct. In addition to targeting the tumor cells, the antibodies also act as a form of immunotherapy, resulting in an enhanced therapeutic effect.

The investigators focused on PD-L1, which is a key target for immune checkpoint inhibition (ICI) immunotherapy. “The hypothesis was that the PD-L1 targeting approach would result in a dual therapeutic role as it both directs the drug loaded Fc-EVs to the tumor and the PD-L1 antibody itself functions as a therapeutic intervention by blocking the immunosuppressive PD1/PD-L1 axis,” they explained.

The studies in mice confirmed that, when given as an injection to animals with breast cancer or melanoma, the treatment reduced tumor growth and improve survival, both at 20 day, and 35 day endpoints. “In fact, this combinational therapy displayed 100% survival at the measured endpoint of 20 days, compared to 35% survival in mock treated mice,” they wrote. When tested with a 35 day endpoint, “… Fc-EV+Dox+PD-L1-Ab treatment again showed a significantly improved disease course with decreased tumor size development over time, and a significantly improved survival.” By the 35 day timepoint median survival of mice treated using the Fc-EV+Dox+PD-L1-Ab nanovesicles had not yet been reached.

The hope is that the new treatment will be more specific and effective in eliminating tumor cells without affecting healthy tissue, compared to current treatment strategies. The researchers plan to investigate whether different combinations of antibodies and drugs can further improve treatment.

“Among other things, we want to investigate the possibility of delivering mRNA as an anticancer drug,” says the study co-author Samir EL Andaloussi, PhD, professor at the department of laboratory medicine, Karolinska Institute. “Ultimately, we hope this can lead to a new treatment platform that can improve treatment efficacy and reduce side effects in difficult-to-treat diseases, especially cancer.”

Writing in their paper, the team concluded, “… the Fc-EV technology offers a combined EV-antibody therapy in which targeting and/or therapeutic antibodies facilitate targeted EV delivery with therapeutic cargo that can generate a synergistic effect.”  In theory, they suggested, the technology could be applied to “a myriad of indications” and used to generate multiple combinations, including classical antibodies, but also, for example, Fc-fused proteins, antibody-drug conjugates or bispecific antibodies. “Subsequent studies on the Fc-EV concept will hopefully explore the therapeutic potential further and possibly translate it to clinical use.”

Scorpius Holdings Expands Its San Antonio Site in San Antonio with New Storage and Testing Facility

CDMO Scorpius Holdings announced a significant expansion of its operational footprint in San Antonio, TX, with the opening of its new storage and testing facility. The expansion increases Scorpius’ warehouse facilities more than sevenfold, from ~3,000 square feet to ~22,000 square feet. The upgrade is in response to the growing demand for Scorpius’ biomanufacturing services and the need to accommodate its expanding client base, according to Jeff Wolf, CEO.

The new facility will feature specialized storage areas with temperatures maintained between -20°C and -80°C for raw materials, alongside spaces designated for contained sampling and quality control testing. Additionally, it will accommodate bulk drug substance storage, ensuring clients’ products are stored under optimal conditions throughout the development and production cycles.

The facility will include a stability program “which will bolster Scorpius’ service offerings by providing in-house stability storage capabilities to foster a smooth transition into onsite testing,” notes Wolf, adding that not only does this add a new dimension to the company’s capabilities, but it also introduces an additional revenue stream.

“These new capabilities enable us to offer our clients continuity through their supply chain and meet their needs from process development through drug substance release,” says Wolf. “This strategic expansion reflects our commitment to excellence and our dedication to supporting our clients’ overall needs.”

Cancer Immunotherapy Hampered by T Cell Inhibitory Checkpoint VISTA Protein

A Cleveland Clinic-led team of scientists and physicians has discovered that the immune checkpoint protein VISTA can directly turn off tumor-fighting T cells during immunotherapy and resist treatment. The team’s preclinical study, including work using mouse models, showed that VISTA can bind to a protein called LRIG1 in T cells, which was previously only thought to promote bone and fat development. When VISTA binds to LRIG1, the researchers found, LRIG1 sends signals that suppress T cell replication, survival, and function.

This interaction can happen between molecules on tumor cells and on T cells, molecules on healthy cells and T cells, and even between molecules on the same T cell. The results suggest that blocking LRIG1 function can halt tumor growth in many cancers.

The findings follow on from another discovery by researchers in the lab of Li Lily Wang, PhD, at the department of translational hematology and oncology, which showed VISTA indirectly suppresses our immune systems by promoting cells called myeloid-derived suppressor cells (MDSCs) that are well known to block T-cell function.

“Our two discoveries combined create a paradigm that explains how VISTA can act as a ‘super villain’ that uses many different weapons to impair antitumor responses during cancer treatments,” said Wang. “This is an insight that drug developers need to consider if they want to boost treatment response rates to their full potential.”

The researchers reported on their latest work in Science Immunology, in a paper titled, “LRIG1 engages ligand VISTA and impairs tumor-specific CD8+ T cell responses.” In their paper, the team concluded, “Here, we identified and characterized a T cell co-inhibitory receptor LRIG1 that interacts with VISTA, an established immune checkpoint protein …These results delineate the role of LRIG1 as an inhibitory immune checkpoint receptor and propose a rationale for targeting the VISTA/LRIG1 axis for cancer immunotherapy.”

Immune checkpoint inhibition (ICI) is a promising approach to cancer therapy, activating tumor immunity and improving the survival of patients, the authors explained. VISTA functions as a negative checkpoint regulator that modulates the immune responses of healthy cells to keep them from attacking our own bodies, protecting the body from autoimmune issues.

However, studies by a group led by Wang have shown that during immunotherapy, VISTA impairs immune activation and prevents T cells from attacking cancer cells. While researchers have worked to generate strategies for blocking VISTA from activation during immunotherapy, success has been limited because exactly how VISTA works hasn’t been known. “VISTA plays an important role in maintaining T cell quiescence,” the team stated, “… however, the molecular mechanisms through which VISTA regulates antitumor immunity are not well defined.” Previous studies, the team noted, “support VISTA as a potent regulator of antitumor immunity,” but its “elusive signaling mechanisms hinder the development of VISTA inhibitors with precise actions and prevent the identification of biomarkers related to VISTA biology.”

As part of their newly reported studies, Wang and colleagues discovered that LRIG1 acts as an inhibitory VISTA receptor. Their initial studies confirmed that LRIG1 was highly expressed in mouse and human T cells after T-cell receptor (TCR) activation. Murine studies indicated that LRIG1 acts as a T-cell inhibitory receptor that mediates the suppressive effects of VISTA in vivo. Further work then provided “strong evidence that LRIG1 impairs the expansion, survival, and effector function of tumor-specific CD8+ CTLs,” the scientists wrote.

When the team investigated LRIG1 expression in human cancer tissues, they found that in human melanoma and endometrial cancer, LRIG1 expression in tumor-associated T cells was correlated with resistance to immunotherapy. The results collectively supported the conclusion that “LRIG1 is a T-cell inhibitor receptor and that elevated LRIG1 expression in CD8+ T cells is associated with resistance to ICI therapies.”

Lead first author Hieu Minh Ta, PhD, said, “Studying the molecular aspect of how LRIG1 functions as VISTA’s receptor on T cells can provide insights on how to successfully block VISTA and improve the clinical outcomes of the patients who don’t respond to existing immune therapies.” Added study co-first author Dia Roy, PhD, “Our findings in human cancer samples inform and support the decision to go after LRIG1 as a potential drug target for new immune checkpoint therapies.”

The newly reported work was carried out as a collaboration between labs led by Wang and Timothy Chan, MD, PhD, chair of the Cleveland Clinic’s Center for Immunotherapy & Precision Immuno-Oncology, director of the Global Center for Immunotherapy and Sheikha Fatima bint Mubarak Endowed Chair in Immunotherapy. The scientists worked closely with physicians, including Brian Gastman, MD, Cleveland Clinic surgical oncologist, and Case Western Reserve University cancer pathologist Stefanie Avril, MD. Gastman and Avril provided the research team with melanoma and endometrial samples for studying the expression of LRIG1.

The lab is now collaborating with many additional clinicians across Cleveland Clinic to learn more about how the VISTA/LRIG1 axis works in regulating immune responses against different cancer types including lung cancer and breast cancer. They expect that combining VISTA-specific inhibitors with existing immunotherapies may reduce resistance and recurrence and improve cancer patient survival.

“The identification of LRIG1 as a VISTA-interacting co-inhibitory receptor in T cells may lead to the development of new cancer therapeutics,” the investigators noted. “ICIs have become breakthrough cancer therapies, but the overall response rate remained suboptimal … LRIG1 expression is detected on TILs from several human cancers and is correlated with resistance to ICI therapies in human melanoma. Therefore, selectively blocking the VISTA/LRIG1 axis may be an effective immunotherapeutic approach for the treatment of cancer.”

HIV Vaccine Triggered Elusive Broadly Neutralizing HIV Antibodies, Trial Shows

An HIV vaccine candidate developed at the Duke Human Vaccine Institute (DHVI) triggered low levels of an elusive type of broadly neutralizing HIV antibodies among a small group of people enrolled in a 2019 clinical trial. The findings are notable for two reasons: 1) the inability to induce B cell lineages of broadly neutralizing antibodies (bnAbs) in humans has been a roadblock to HIV vaccine development and 2) bnAbs take years to develop in people living with HIV-1.

The finding not only provides proof that a vaccine can elicit these antibodies to fight diverse strains of HIV, but also that it can initiate the process within weeks, setting in motion an essential immune response.

The vaccine candidate targets the area on the HIV-1 outer envelope known as the membrane proximal external region (MPER). More specifically, the candidate includes a “peptide/liposome immunogen targeting B cell lineages of HIV-1 envelope (Env) membrane-proximal external region (MPER) bnAbs.” Antibodies against this region—which remains stable even as the virus mutates—in the HIV outer coat can block infection by many different circulating strains of HIV.

This work is published in Cell in the paper, “Vaccine induction of heterologous HIV-1-neutralizing antibody B cell lineages in humans.

“This work is a major step forward as it shows the feasibility of inducing antibodies with immunizations that neutralize the most difficult strains of HIV,” said Barton F. Haynes, MD, director of the DHVI. “Our next steps are to induce more potent neutralizing antibodies against other sites on HIV to prevent virus escape. We are not there yet, but the way forward is now much clearer.”

The research team analyzed data from a Phase I clinical trial, the HVTN 133 clinical trial. Twenty healthy, HIV-negative people enrolled in the trial. Fifteen participants received two of four planned doses of the investigational vaccine, and five received three doses.

After two immunizations, the vaccine had a 95% serum response rate and a 100% blood CD4+ T-cell response rate. Most of the serum responses mapped to the portion of the virus targeted by the vaccine. Broadly neutralizing antibodies were induced; the most potent of which neutralized 15% of global tier 2 HIV-1 strains and 35% of clade B strains. In addition, the authors noted that “neutralization was enhanced by vaccine selection of improbable mutations that increased antibody binding to gp41 and lipids.”

The trial was halted when one participant experienced a non-life-threatening allergic reaction. The team investigated the cause of the event, which was likely from an additive.

“To get a broadly neutralizing antibody, a series of events needs to happen, and it typically takes several years post-infection,” said Wilton Williams, PhD, an associate professor in Duke’s department of surgery and a member of DHVI. “The challenge has always been to recreate the necessary events in a shorter space of time using a vaccine. It was very exciting to see that, with this vaccine molecule, we could actually get neutralizing antibodies to emerge within weeks.”

Other features of the vaccine were also promising, most notably how the crucial immune cells remained in a state of development that allowed them to continue acquiring mutations, so they could evolve along with the virus.

The researchers said there is more work to be done to create a more robust response, and to target more regions of the virus envelope. A successful HIV vaccine will likely have at least three components, all aimed at distinct regions of the virus.

“Ultimately, we will need to hit all the sites on the envelope that are vulnerable so that the virus cannot escape,” Haynes said. ”But this study demonstrates that broadly neutralizing antibodies can indeed be induced in humans by vaccination. Now that we know that induction is possible, we can replicate what we have done here with immunogens that target the other vulnerable sites on the virus envelope.”

bone scans
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The new Scorpius facility in San Antonio will feature specialized storage areas with temperatures maintained between -20°C and -80°C for raw materials, alongside spaces designated for contained sampling and quality control testing. Additionally, it will accommodate bulk drug substance storage, ensuring clients' products are stored under optimal conditions throughout the development and production cycles. A stability program is expected bolster Scorpius' service offerings as well.
Scientists discovered that the immune checkpoint protein VISTA can turn off tumor-fighting T cells during immunotherapy. Preclinical studies in mice and in human tissue showed VISTA binds to the LRIG1 receptor, resulting in suppression of T cell replication, survival and function, and correlating with treatment resistance. The combined results of this and the team’s previous studies creates “a paradigm that explains how VISTA can act as a ‘super villain’ that uses many different weapons to impair antitumor responses during cancer treatments.”
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An HIV vaccine candidate developed at the Duke Human Vaccine Institute triggered low levels of an elusive type of broadly neutralizing HIV antibodies among a small group of people enrolled in a 2019 clinical trial. The finding provides proof that a vaccine can elicit these antibodies to fight diverse strains of HIV and that it can initiate the process within weeks, setting in motion an essential immune response.