As part of their effort to answer a decades-old biological question about how the hepatitis B virus (HBV) can establish an infection in liver cells, researchers led by teams at Memorial Sloan Kettering Cancer Center (MSK), Weill Cornell Medicine, and the Rockefeller University have identified a vulnerability that may open the door to new HBV treatments.

The team successfully disrupted the virus’s ability to infect human liver cells in the laboratory using a chromatin-destabilizing molecule CBL137, which is already in clinical trials against cancer. The results lay the groundwork for animal model studies and potential drug development.

Headed by chemical biologist Yael David, PhD, at MSK, working with hepatologist and virologist Robert Schwartz, MD, PhD, at Weill Cornell Medicine and Viviana Risca, PhD, at the Rockefeller University, the researchers reported on their findings in Cell, in a paper titled, “A nucleosome switch primes hepatitis B virus infection.”

Chronic HBV infection is an incurable pathogen responsible for causing liver disease and hepatocellular carcinoma, the authors wrote. “Over 325 million people worldwide are chronically infected by hepatitis B virus … leading to almost one million deaths annually despite the availability of effective vaccines.” Chronic infection leaves patients at risk for advanced liver disease, and HBV is estimated to cause nearly half of all hepatocellular carcinoma cases, the team continued.

The newly reported research began with a chance meeting and a longstanding paradox. Schwartz, an associate professor of medicine in the Division of Gastroenterology and Hepatology at Weill Cornell Medicine, was introduced to David about six years ago at a retreat for Weill Cornell Physiology, Biophysics, and Systems Biology graduate school faculty, where they both hold appointments.

“On the surface, our research programs seem to have no overlap,” David said. “He studies hepatitis B, while my lab focuses on understanding how gene expression is regulated through a process called epigenetics. However, I was fascinated to discover that viruses like hepatitis B hijack epigenetic mechanisms, even using human DNA-packaging proteins to regulate their activity.”

Not long after, study first author Nicholas Prescott, PhD, then a doctoral student in the Tri-Institutional PhD Program in Chemical Biology, was preparing for a stint in the David Lab at MSK’s Sloan Kettering Institute. “His interest in epigenetic regulation in pathogens immediately made me consider HBV an ideal model system for him to explore,” David said.

At the heart of the mystery that intrigued the researchers lies a key viral gene that encodes for the HBV X protein (HBx). This protein is essential for HBV to establish a productive infection in host cells and the expression of its viral genes. However, the X gene itself is encoded within the viral genome.

“This raises a classic chicken-and-egg question that has puzzled scientists for decades,” David said. “How does the virus produce enough X protein to drive viral gene expression and establish infection?” The authors further commented “… HBx is absent from the virion and must be expressed de novo in freshly infected hepatocytes …”

Furthermore, the gene that encodes protein X is considered the virus’s oncogene—that is, the gene responsible for the disease’s progression toward cancer, Prescott added. That’s because protein X degrades proteins in the host that are involved with DNA repair. Not only does this keep the host from silencing protein X’s activity, but the infected cells are also more likely to accumulate DNA errors that build up over the years and decades, leading to the development of cancer.

“One of the main challenges with treating hepatitis B is that the existing treatments can stop the virus from making new copies of itself, but they don’t fully clear the virus from infected cells, allowing the virus to persist in the liver and maintain chronic infection,” noted Schwartz, whose lab contributed biological and clinical expertise in the virus, as well as the human liver cell models used in the study. “The current standard of care (long-term treatment with oral nucleos(t)ide analogs or short-term treatment with interferon-alpha injections) halts viral replication but falls short of eradicating cccDNA in infected hepatocytes, allowing the minichromosome to persist and sustain chronic infection,” the investigators stated. “Even with long-term antiviral treatment, basal levels of the HBV oncogenic X protein (HBx) remain in hepatocytes to promote genomic instability and disease progression.”

The hepatitis B vaccine is effective, but maintaining immunity often requires booster shots. Moreover, it doesn’t help people who are already infected. This happens, for example, due to transmission of the virus from mother to child, which is very common in developing countries. Access to vaccines and treatment is also more limited in some parts of Africa and Asia, where rates of infection are higher.

Digging into the mystery of protein X was a challenge, explained Prescott, who is now a postdoctoral fellow in the Laboratory of Chromosome and Cell Biology at the Rockefeller University. The existing tools weren’t capable of shedding light on what was happening in those critical early hours of an infection.

Early in infection, HBV establishes an independent “minichromosome” consisting of the viral covalently closed circular DNA (cccDNA) genome and host histones. But as the authors noted, “Despite extensive study, a significant knowledge gap remains regarding the role of viral chromatin status in establishing infection.”

The David Lab’s expertise is in how DNA gets packaged, read, and modified proved essential. For their newly reported research, the researchers successfully generated the HBV minichromosome for the first time, using their capabilities in reconstituting viral DNA in complex with human histones—the proteins that package and organize DNA. The research team determined that in order for protein X to get made, the hepatitis B virus’s DNA needs to get organized into DNA-histone complexes called nucleosomes. “… we generated recombinant, chromatinized cccDNA, allowing us to characterize its biophysical properties and to map nucleosome positioning on the minichromosome,” the scientists explained.

“This platform became a powerful tool not only to study the virus’s biochemistry but also to analyze, in detail, what happens in the critical first hours of an infection,” David said.

Nucleosomes are like beads on a string—the string is the viral DNA, and the beads are host-provided histone proteins, around which DNA gets wrapped; nucleosomes are the building blocks of chromatin, the material that makes up chromosomes.

It was this part of the project that tapped into the expertise of Risca at Rockefeller University. The Risca Lab studies the 3D architecture of the genome and how the packaging of DNA helps to control the transcription of genes. They had the tools and expertise to ensure that what the scientists were seeing in the new platform for studying the virus matched the reality of a human infection.

“Conventional wisdom says that packaging a gene’s DNA into nucleosomes would block or slow down the cell’s ability to read out that gene to make functional proteins, like protein X,” Risca commented. “But in complex organisms like humans and in the viruses that infect us, gene regulation is not always so straightforward. The presence and the positioning of nucleosomes on DNA can be important in directing cellular mechanisms to transcribe some genes. We found that to be the case for the HBV gene encoding protein X—the presence of nucleosomes on the viral genome is necessary for the transcription of RNA that gives rise to functional protein X.” The authors added. “Altogether, these data suggest a correlation between early cccDNA chromatinization and X transcription, which both occur during the first hours of infection.”

This discovery opens the door to understanding how the X gene is regulated and how HBV infection is established. Moreover, the researchers discovered a potential therapeutic opportunity. If it’s possible to disrupt the formation of these chromatin structures, then this could disrupt the virus’s ability to start and maintain an infection. “Given this link between chromatinization and transcription, we next hypothesized that disrupting chromatin assembly might inhibit viral transcription,” they wrote.

The team tested five small-molecule compounds known to impair chromatin formation. Only one, an anticancer drug candidate called CBL137, blocked production of protein X in liver cells.

Importantly, the compound worked in vitro at very low concentrations—many times smaller than participants in clinical trials for cancer were receiving, and using doses that only affected the virus, but not human cells. Their experimental results, they noted, “… demonstrate CBL137 as an effective inhibitor of HBV transcription and replication that may pose a potential therapeutic avenue to treat infections.”

David noted, “This made us very optimistic about the possibility of developing a treatment approach while preventing or limiting side effects. Moreover, if these results are confirmed through additional study, we are optimistic the approach could be used to treat chronic infections for the first time—and therefore could represent a potential cure.”

Additionally, CBL137 might prove similarly useful to target or study other chromatinized DNA viruses such as herpesviruses and papillomaviruses, the researchers noted. “Chromatin destabilization by CBL137 merits further investigation to test if it could prove similarly effective on other pathogens. Indeed, recent studies reported encouraging results for CBL137 as a latency reversing agent in HIV-1 infection and even a lead for drug discovery efforts against human African trypanosomiasis, underscoring its potential as a therapeutic against infectious diseases.

The project David pointed out, “…started from our fundamental interest in how the virus’s chromosomes might look and function and led to unexpected discoveries of how the viral infection is established in human cells.”

To further develop the team’s research toward a potential clinical trial, the next step would be to study the safety and effectiveness of CBL137 in animal models—though these are limited due to the narrow range of species HBV can infect, the researchers said.

All of the researchers stressed that the study wouldn’t have been possible without the close collaboration between the three institutions, which brought together the necessary expertise and technological resources—from MSK’s atomic force microscope to the Genomics Resource Center and High-Performance Computing Cluster at Rockefeller University.

Prescott commented, “This is a great example of how investment in ‘basic science’ and investigation of fundamental biological questions can open the door to medical advances,” he said. “I always thought I’d be working on questions that decades later someone might cite in a paper when they come up with a cure for some disease. Never in a million years did I expect to lead a project that identified such a strong candidate for drug development for a global scourge like hepatitis B.”

Atrandi Biosciences has raised $25 million in a Series A funding round led by Lux Capital, with participation from Vsquared Ventures, Practica Capital, Metaplanet, and CRIDS Capital. The company plans to use the funds to develop new products based on its semi-permeable capsule (SPC) technology as well as to set up an office in Boston which will allow the company to better serve its U.S. customer base.

Atrandi, which means “you discover” in Lithuanian, was launched in 2016 to address technological challenges associated with single-cell analysis. As Juozas Nainys, PhD, Atrandi’s CEO & co-founder, explained, the company was launched “to bridge a fundamental gap in biological research—the need for high-throughput, scalable technologies to manipulate and analyze single cells with precision.” Furthermore, “our SPC technology is a fundamental breakthrough born from a need to overcome the limitations of existing single-cell analysis tools, giving researchers the possibility to generate rich datasets with an unprecedented combination of throughput, multimodality and data quality.”

Atrandi will use some of the funds from its Series A to extend its offerings for DNA analysis from single cells, Nainys told GEN. The company already has a solution for microbial cells and expects to launch an option that works for eukaryotic cells later this year. They will be able to support whole genome analysis as well as more targeted analysis of single cells. Next year, Atrandi will focus on developing products for multi-omic analysis—specifically DNA and RNA analysis from single cells.

Designed for high-throughput single-cell research, Atrandi’s SPCs are aqueous compartments that are enclosed by a semi-permeable shell. They are designed to isolate single cells and nucleic acids while enabling the exchange of small molecules like enzymes and nutrients. The ability to exchange materials is an important part of SPCs value proposition for single cells and something that sets Atrandi’s technology apart from current droplet microfluidics technologies, according to Nainys. Typically, “once you form a droplet you can add reagents to it … but you can never remove [them],” he explained. “That’s very limiting [because] there are so many different molecular biology reactions that just do not work together or require specific pH [and] buffers.”

Nainys interest in single-cell technologies predates Atrandi’s founding. During his PhD, he worked in a laboratory focused on developing single-cell RNA sequencing technologies. “The single-cell revolution was really brought about by droplet microfluidics,” he said. “The lab that I joined specialized in droplet microfluidics and as experts in that particular technology, we saw that there are … a lot of things that can not be done in droplets.” That led him and Atrandi’s co-founders to launch the company in 2016 with an eye toward developing and commercializing SPCs as well as instruments for generating them.

To date, Atrandi has released three products based on its technology. The first of these is the Flux Microfluidic Device, a user-friendly system designed for high-throughput single-cell isolation into SPCs. The primary goal of this system was to “make the compartmentalization of the sample as seamless as possible so it’s really a push button,” Nainys said. “It’s more for a broader audience that doesn’t want to tinker with the technology.”

The two other products in Atrandi’s portfolio are the Onyx Droplet Generator, a microfluidic platform designed for high-throughput single-cell and single-molecule applications, and the Styx High-Throughput Screening Platform, which is designed for fluorescence-activated droplet sorting. These platforms are designed for users who want more control of their workflows and the ability to adjust different parameters as needed, Nainys said. They are compatible with SPCs as well as droplets. Additionally, “we also worked hard to make sure that the capsules integrate well with any readout method” including sequencing instruments, microscopy, and mass spectrometers.

So far, Atrandi has installed over 150 devices in labs in Europe, North America, and South Korea where they are used for a wide range of applications. About half of its customers are based in U.S. laboratories. People are using the solutions in different ways including studies in oncology, immunology, and microbiology, Nainys said. “It really is all over the map but at the end of the day it’s analyzing single cells as part of a complex biological system.”

Researchers at Wuhan University have discovered a cholesterol metabolite that may play a critical role in the development of Parkinson’s disease (PD). Studies in mice, led by Zhentao Zhang, MD, PhD, indicated that the metabolite, 24-hydroxycholesterol (24-OHC), promotes the spread of Lewy bodies and the death of dopaminergic neurons in the brain, the two major hallmarks of Parkinson’s disease. 24-OHC is produced from cholesterol by the actions of an enzyme, cholesterol 24-hydroxylase (CYP46A1), and the researchers suggest that blocking the activity of 24-OHC or preventing the metabolite from being produced could potentially represent effective strategies for treating Parkinson’s disease.

Zhang and colleagues reported on their findings in PLOS Biology, in a paper titled, “The cholesterol 24-hydroxylase CYP46A1 promotes α-synuclein pathology in Parkinson’s disease.”

PD is the second most common neurodegenerative disease, and leads to slowness of movement, tremor, rigidity, cognitive impairment, and neuropsychiatric symptoms, the authors explained. The disease is characterized by the formation of Lewy bodies composed of aggregates of α-synuclein (α-Syn), and death of dopaminergic neurons in the substantia nigra.

“Converging lines of evidence indicate that α-Syn fibrils can spread in a prion-like manner in the brain, leading to self-propagation and cell-to-cell transmission of protein aggregate,” the investigators stated. “Although it is clear that α-Syn aggregation underlies the pathology of PD, what drives the spread of α-Syn remains unclear.”

Their newly reported study was designed to investigate what causes the spread of pathological α-Syn, with the authors hypothesizing that the culprit is the cholesterol metabolite 24-OHC, which is known to present at high levels in the brains of people with Parkinson’s disease, and which increases with age. “Various types of clinical evidence indicate that the levels of 24-OHC in the cerebral spinal fluid (CSF) are increased in PD patients and are correlated with the duration of the disease,” they wrote, also commenting that “previous studies demonstrate cholesterol 24-hydroxylase (CYP46A1) increases the risk for PD.”

Through their study, the team first confirmed that 24-OHC levels were higher in the blood of patients with Parkinson’s disease as well as in a mouse model of the disease. The results, they wrote, “… suggest that both CYP46A1 and 24-OHC increase in an age-dependent manner and are elevated in PD patients, PD model mice, and aged wild-type mice.”

The researchers then blocked 24-OHC production in the mouse model by knocking out the CYP46A1 enzyme. This reduced both the spread of the harmful α-Syn fibers and damage to the dopamine neurons in the critical part of the brain. “… CYP46A1 knockdown relieves the spread of α-Syn pathology and the loss of dopaminergic neurons,” they commented.

Further experiments with neurons cultured in a dish showed that the addition of 24-OHC caused normal α-Syn to change into harmful α-Syn fibers. Injecting mice with these fibers led to a greater spread of Lewy bodies, more dopaminergic neuron degeneration, and greater motor deficits than did injecting them with α-Syn fibers formed in the absence of 24-OHC. The researchers suggested that drugs that prevent cholesterol from being converted to 24-OHC might therefore be an effective treatment for the disease.

The authors suggest that their findings “… indicate that the cholesterol 24-hydroxylase CYP46A1 plays a pivotal role in the progression of α-synuclein pathology in Parkinson’s disease, highlighting its potential as a therapeutic target for Parkinson’s disease.” In their report, they further stated, “It will be interesting to determine whether CYP46A1 and 24-OHC can serve as theranostic biomarkers for disease-modifying therapies.”

In a new study published in GEN’s sister peer-review journal, GEN Biotechnology, titled, “Exploring Structure-Function Relationships in Engineered Receptor Performance Using Computational Structure Prediction,” researchers from Northwestern University present a new structure-based analysis to guide the design of synthetic receptors for improved cell therapies.

“A key challenge is that subtle structural changes can have profound functional consequences, and thus cell therapies can benefit from improved methods for tuning receptor functions at the structural level,” said Joshua Leonard, PhD, professor of chemical and biological engineering at Northwestern University and corresponding author of the study, in an interview with GEN.

In cell therapies, engineered receptors need to encompass both safety and efficacy, such as maintaining a resting state until it encounters a disease target. However, the structure of these receptors is generally poorly understood, which can have a substantial impact on therapeutic performance. In the example of CAR T-cell therapies, structurally tuning receptors can avoid tonic signaling, or activation in the absence of the target stimulus, which often drives T-cell exhaustion and diminishes the therapy’s efficacy against targets, such as a tumor. 

The authors generated receptor structural models to investigate the structure-function relationships using a case study that characterized the conversion of natural human cytokine receptors into engineered receptors. They hypothesized that structure prediction might be most feasible on a dataset based on natural receptors, given the reliance of existing tools on sequence alignment with known proteins. 

The authors also extended the analysis to “categorical” variables that describe the identity of specific domain choices and the way in which they are combined, which implicitly captures properties that are unique to the individual chains and not described by structural features directly, such as protein expression, membrane localization, protein stability. Overall, they observed structure-function trends that were largely conserved across structurally diverse receptor sets.

Engineered receptors are a particularly challenging class of proteins to structurally define, as they are relatively large, contain a difficult-to-characterize hydrophobic transmembrane domain, and can adopt multiple conformations. Groundbreaking tools, such as AlphaFold, which solved biology’s hallmark problem of determining a protein’s 3D structure from its sequence and earned DeepMind’s Demis Hassabis, PhD, and John Jumper, PhD, a share of the 2024 Noble Prize in Chemistry, AlphaFold Multimer, and RoseTTAFold (developed by fellow 2024 Nobel Laureate, David Baker, PhD), have been able to construct complete models of natural single-pass transmembrane receptors. However, distilling which structural features impact receptor performance remains an ongoing gap.

“For all the aspects of the receptor we don’t understand, we have to spend more time and effort empirically exploring design choices, and even when good designs are identified, this lack of structural knowledge makes it more difficult to understand why certain choices yield good performance,” said Leonard.

The GEN Biotechnology study proposes potential design rules that are experimentally testable and can guide future engineering across receptor families. Leonard and William Corcoran, lead author of the study and a PhD candidate at Northwestern, expect that these insights will most immediately benefit the design of synthetic receptors that share a similar signaling mechanism, given that the study was based on the conversion of natural receptors (which employ multiple mechanisms) into synthetic receptors (which employ a single mechanism).

Looking ahead, the authors stated that a natural evolution of this workflow is to generate libraries that explore greater design space. In addition, Leonard’s group has several ongoing projects for developing and employing distinct synthetic receptor families and plans to apply the insights from this study to design new synthetic receptors for more applications.

“Ultimately, this study makes us optimistic that protein structure prediction tools, while ever improving, are already useful for making plausible connections between receptor sequence and receptor function, and that fact should accelerate the development of