Characterizing clonal evolution in blood cancers like acute myeloid leukemia (AML) is critical for understanding their mutational histories and how cell populations change during disease development.

In this GEN webinar, Robert Bowman, PhD, will discuss his lab’s approaches for modeling clonal evolution in mouse models of disease. His group deploys multi-recombinase models to study the stepwise acquisition of mutations seen in AML. These approaches allow for the evaluation of how mutation order impacts disease development. They have characterized the hierarchy of cellular differentiation using flow cytometry and single cell RNA sequencing, recently integrating the ScaleBio Single Cell RNA Kit into their workflow. He will discuss a specific study focusing on FLT3-mutant AML, present data comparing genetic deletion versus chemical inhibition with FDA-approved tyrosine kinase inhibitors, and finally, his plans to further deploy models of oncogene-dependency.

A live Q&A session will follow the presentation, offering you a chance to pose questions to our expert panelist.

Webinar produced with support from:

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Following their success against COVID-19 infections, interest in RNA-based therapeutics is growing rapidly. And as demand for RNA drugs continues to grow and “additional products come to market, we will exceed the current global supply of acetonitrile, the organic solvent used in chemical RNA synthesis methods,” said co-first author Jonathan Rittichier, PhD, a former postdoctoral fellow at the Wyss and HMS. 

Besides reducing the creation of toxic synthesis byproducts, the technology that Rittichier and his collaborators, which includes George Church, PhD, Wyss core faculty member and HMS professor of genetics, have developed incorporates common molecular modifications found in RNA drugs today and can be used with novel RNA chemistries to develop new therapies. “Delivering RNA drugs to the world at these scales requires a paradigm shift to a renewable, aqueous synthesis, and we believe our proprietary enzymatic technology will enable that shift,” Rittichier said.

The starting point for the technology is an enzyme from Schizosaccharomyces pombe, a strain of yeast, that is used to form RNA strands. The scientists engineered a more efficient version of the enzyme that can also incorporate nonstandard nucleotides into RNA—a necessary improvement since every FDA-approved RNA drug includes modified nucleotides that increase their stability in the body or equip them with new functions. Their approach also uses milder methods to remove protecting groups which are added to nucleotides to protect them during the synthesis process. 

Furthermore, the researchers modified the nucleotides by attaching a chemical group called a “blocker” that ensures that nucleotides are added to the RNA chain one at a time. Once the desired nucleotide has been added to the link, the blocker is removed to allow the next nucleotide in the sequence to bind. This two-step process is simpler and uses fewer reagents than the typical four-step chemical synthesis. According to the scientists, their process is also 95% efficient and can build RNA molecules as long as 23 nucleotides. However, they noted that “overall yields in bulk phase were lower than those in standard RNA oligonucleotide synthesis” likely due to “multiple rounds of purification after both extension and deblocking.”

“Enzymatic nucleotide synthesis technologies offer many advantages as an alternative to chemical-based methods,” Church said in a statement. “This platform can help unlock the immense potential of RNA therapeutics in a sustainable way, especially manufacturing high-quality guide RNA molecules for CRISPR/Cas gene editing.” 

Rittichier, Church, and others launched EnPlusOne Biosciences in 2022 to commercialize the technology with funding from Northpond Ventures, Breakout Ventures, and Coatue. The company is currently using the technology to manufacture small interfering RNAs that could be used to treat various diseases. 

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Kappler said the bacterium had a unique ability to “talk” to and deactivate the immune system, convincing it there was no threat. “These bacteria are especially damaging to vulnerable groups, such as those with cystic fibrosis, asthma, the elderly, and Indigenous communities,” Kappler said. “In some conditions, such as asthma and chronic obstructive pulmonary disease, they can drastically worsen symptoms. Our research shows the bacterium persists by essentially turning off the body’s immune responses, inducing a state of tolerance in human respiratory tissues.”

The researchers reported on their studies, and results, in PLOS Pathogens, in a paper titled “Tolerance to Haemophilus influenzae infection in human epithelial cells: Insights from a primary cell-based model.”

Respiratory tract infections are highly debilitating, and Haemophilus influenzae is a bacterial pathogen that is associated with persistent acute and chronic respiratory tract infections, particularly among vulnerable individuals, the authors explained. “Haemophilus influenzae is a human-adapted pathobiont that inhabits the nasopharynx as a commensal but causes disease in other parts of the respiratory tract.” What are classed as nontypeable strains of H. influenzae (NTHi) are the most common type of clinical isolate, the team continued. In addition to causing acute diseases such as pneumonia, these strains are a major cause of exacerbations of chronic lung diseases, including in patients recovering from COVID-19. Interactions between the bacteria and the respiratory epithelia represent a key factor in NTHi virulence,” the team continued. “Despite this, insights into the molecular interactions that allow NTHi to persist in contact with human epithelia are lacking, but likely hold the key to uncovering both bacterial and host processes that are crucial for infection.”

For their newly reported studies the team generated primary normal human nasal epithelia (NHNE), derived from cells from five healthy donors, and monitored the effects on tissue gene expression of NTHi infection. They first prepared the human nasal tissue in the lab, growing it to resemble the surfaces of the human respiratory tract. They then monitored post infection (p.i.) gene expression changes over a 14-day period of “infection” with the NTHi. “Persistent infections rely on close molecular interactions between the human respiratory cells and the bacterial pathogen,” the team noted, “… and here we have investigated changes in host and bacterial cells during persistent, long-term infections with H. influenzae.”

Their found very limited production of inflammation molecules over time, which normally would be produced within hours of bacteria infecting human cells. “We then applied both live and dead Haemophilus influenzae, showing the dead bacteria caused a fast production of the inflammation makers, while live bacteria prevented this,” professor Kappler said.  “This proved that the bacteria can actively reduce the human immune response.”

In their paper the authors wrote, “… Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H. influenzae infection with an initial, ‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently, an apparent tolerance to large extracellular and intraepithelial burdens of H. influenzae developed, with NHNE transcriptional profiles resembling the pre-infection state.”

This is the first time that large-scale, persistence-promoting immunomodulatory effects of H. influenzae during infection have been observed, they stated. “In addition to providing first molecular insights into mechanisms enabling persistence of H. influenzae in the host, our data further indicate the presence of infection stage-specific gene expression modules, highlighting fundamental similarities between immune responses in NHNE and canonical immune cells, which merit further investigation.”

Co-author and pediatric respiratory physician emeritus professor Peter Sly, MD, at UQ’s Faculty of Medicine, said the results show how Haemophilus influenzae can cause chronic infections, essentially living in the cells that form the surface of the respiratory tract.

“This is a rare behavior that many other bacteria don’t possess,” professor Sly said.
“If local immunity drops, for example during a viral infection, the bacteria may be able to ‘take over’ and cause a more severe infection.” The findings will lead to future work towards new treatments to prevent these infections by helping the immune system to recognize and kill these bacteria. “We’ll look at ways of developing treatments that enhance the immune system’s ability to detect and eliminate the pathogen before it can cause further damage,” Kappler added.

In their paper the authors concluded, “… our data provide first evidence that NTHi infections can delay strong inflammatory responses in human epithelia and induce an apparent tolerance of NTHi infection that had not been previously observed, but could be a driver of NTHi persistence in the human respiratory tract. This state of ‘peaceful’ coexistence of NHNE and NTHi required infection with live NTHi, which indicates an active immunomodulatory role for NTHi.”

They pointed out that further research is needed to investigate whether bacterial effector proteins or metabolites are involved in triggering NHNE tolerance of NTHi infection, and what mechanisms within human epithelia cause differences in tolerance of NTHi infections.

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These so-called microproteins or noncoding ORF-derived (ncORF) peptides are small proteins expressed only by cancer cells that can be used to activate the immune system against cancer. Furthermore, these molecules are generated by genes that were once considered incapable of encoding proteins. The scientists discovered the proteins in this study by integrating and analyzing information from tumor and healthy tissue collected from over a hundred patients with hepatocellular carcinoma including RNA sequencing, immunopeptidomics, and ribosome profiling data. 

There is significant interest in cancer vaccines which rely on the immune system’s ability to recognize foreign proteins generated as a result of mutations in cancerous cells. The challenge lies with cancers with low mutation rates like liver cancers. Microproteins could be a solution in these cases. The results reported in the paper highlight the potential of using microproteins exclusively expressed in tumor cells as targets for new treatments. Specifically, the researchers identified “a subset of 33 tumor-specific long noncoding RNAs expressing novel cancer antigens shared by more than 10% of the HCC samples analyzed, which, when combined, cover a large proportion of the patients,” according to the paper.

In fact, “we have seen that some of these microproteins can stimulate the immune system, potentially generating a response against cancer cells,” said Puri Fortes, PhD, one of the paper’s authors and a researcher at CIMA as well as the Network Biomedical Research Center for Liver and Digestive Diseases (CIBERehd). According to the paper, when the team tested four ncORF-derived peptides in transgenic mice, they found that two of them could generate a significant immune response involving CD8+ T cells.  “This response can be enhanced with vaccines, similar to the coronavirus vaccines, but producing these microproteins. These vaccines could stop or reduce tumor growth,” said Fortes. 

Also, unlike other types of vaccines based on patient-specific mutations, a potential anticancer vaccine that targets ncORF peptides could be used to treat multiple people, as the same microprotein is expressed in various patients, the researchers noted. 

The post Liver Tumor Microproteins Could Be Key to Developing New Cancer Vaccines appeared first on GEN - Genetic Engineering and Biotechnology News.

The work, which is published online today in Nature in the paper “In situ targeted base editing of bacteria in the mouse gut,” was a team effort by the scientists at Eligo, led jointly by Jesus Fernandez-Rodriguez, PhD, (Eligo’s VP of Technology), David Bikard, PhD, (Eligo co-founder and head of the Synthetic Biology Group at Institute Pasteur), and Xavier Duportet, PhD, (Eligo’s CEO and chairman).

Before this report, notes Bikard, “it was still an outstanding question whether it would even be possible to genetically modify a whole target bacterial population in an animal. There could have been fundamental barriers that would’ve made this impossible. But here we show that we can do it!”

Eligo’s advance, which combined research in both vector engineering and payload modification, is exciting on two fronts: it could open the door to new microbiome genome-editing therapeutic modalities. And it launches a microbiome-editing toolbox that has been previously unavailable.

This work is “ushering in a new era of microbiome engineering,” notes Rodolphe Barrangou, PhD, professor at North Carolina State University and editor-in-chief of The CRISPR Journal. “This proof-of-concept study is just not for E. coli or the mouse gut microbiome; it can be used much more widely, for all kinds of things, and can be deployed at scale.”

Brady Cress, PhD, principal investigator of microbiome editing technologies at the Innovative Genomics Institute at the University of California, Berkeley, agrees. Cress told GEN that this is “a massive step forward that opens the door to rewriting our microbiomes for optimal health.”

Tweaking, not altering

Duportet co-founded Eligo with Bikard a decade ago; the two friends were still in training when they had the idea for the company—Duportet was a graduate student at MIT, Bikard a postdoctoral fellow in Luciano Marraffini PhD’s lab at the Rockefeller University. (Marraffini and Tim Lu, MD, PhD, are Eligo’s other scientific co-founders.) Today, Duportet and Bikard are a dynamic duo—with Duportet at the helm of the company and Bikard a scientific advisor, who remain close friends while collaborating scientifically.

Current microbiome approaches are typically based on altering the compositions of the bacteria. The idea is to introduce bacterial species to change the balance (like probiotics) or to remove others. Eligo’s focus here was different. The idea is not to kill the bacteria but rather, as Duportet explains, to “inactivate its pathogenic potential and leave the bacteria in place.”

“If you are trying to target bacteria that has a niche,” notes Bikard, “completely removing it from the niche might be very challenging. Unless there is something else there to take its place, it will just grow back. So, it is a better strategy to disarm it, rather than kill it.”

Two-fold

Eligo’s new data are not the first to demonstrate editing of the microbiome in vivo, however. In May 2023, research from the Danish company SNIPR Biome was published in Nature Biotechnology in a paper entitled, “Engineered phage with antibacterial CRISPR–Cas selectively reduce E. coli burden in mice.” In it, the researchers identified eight phages (after screening a library of 162 phages) that delivered a CRISPR-based gene-editing payload that resulted in a reduction of E. coli in the mouse gut. In the SNIPR Biome study, the E. coli were killed by CRISPR.

It has been known for a decade—since Bikard and Marrafini’s Nature Biotechnology paper in 2014—that cutting the bacterial chromosome with CRISPR-Cas kills bacteria efficiently. But the first generation of gene-editing tools were not very efficient tools, notes Bikard. They could be used in a lab setting to make modifications but most of the bacteria would be killed in the process. Therefore, if the goal is to make modifications in vivo, and maintain the bacterial population, the first generation of CRISPR tools for bacteria “would not cut it.”

The game changed when base editing—a more precise form of genome editing developed in the Broad Institute lab of David Liu, PhD—entered the toolbox. Eligo worked to bring together their knowledge in genome editing and delivery to allow for efficient editing of bacteria without changing the composition of the microbial population.

When targeting E. coli strains colonizing the mouse gut, Eligo’s technology modified the target gene in more than 90% of the bacteria, reaching up to 99.7% in some cases. These modifications remained stable for at least 42 days.

Barrangou notes that the penetrance of the edits showed remarkable efficiency. “They are setting the bar,” he told GEN. “Being able to do it is one thing. But being able to do it with that kind of longevity and efficiency is practically important and sets the stage for new opportunities in the field.”

For Bikard, working out the gene editing was not as big a hurdle as the delivery. Eligo modified the site fibers on a phage chassis (from phage lambda) to target specific bacterial strains. The phage vector can be modified to target different bacterial strains or species with additional engineering.

Cress thinks of it as “a reprogrammable platform” for targeting different bacteria. That said, while this research provides an impressive blueprint using the most well-studied phage-bacteria pair, Cress notes that expanding it to other microbes will necessitate developing efficient genetic tools for non-model bacteria and a deeper understanding of the genetics and biology of less well-studied phages.

Another advance, Duportet notes, is that his team was able to demonstrate the same efficiency of editing using a non-replicative plasmid in the target bacteria. This is an additional benefit because they don’t maintain a transgene in the microbiome of the animals.

Long road ahead

The long-term goal at Eligo is to develop therapeutics—not necessarily for infectious diseases. The interest extends to those that would change the genetic content of the microbiome that alter a factor of host–focused diseases.

One example where this could be applicable is the delivery of a base editor to commensal intestinal E. coli that express the toxin colibactin, to inactive its mutagenic potential, therefore preventing the progression of human colorectal tumors.

But there is a long road ahead and challenges remain. One, notes Cress, is that this approach uses short-lived delivery of gene editing machinery to make gene disruptions, but other types of edits like gene insertions take longer to write (e.g. CRISPR-associated transposases) and thus will likely require different delivery approaches. Another point Cress considers is that “the type of edits made in this study could potentially revert through natural mutation, making gene removal a more durable solution than gene disruption.”

This study also raises new questions. Teasing apart the genetic network of the microbiome is in its infancy. Do researchers have enough knowledge to use this new tool?

“It’s important to have the genome editing tools,” notes Barrangou. But in the end, “what really matters is knowing what to target. Knowing what to target and what edit you want is part of the secret sauce.”

But Bikard reckons that this work will help answer some of those questions. This will be an extraordinary tool for researchers, he says, because it offers the possibility to probe gene function directly in the animal. He is excited to use it in his academic lab on the other side of Paris from Eligo’s base.

Duportet hopes that scientists will use the method and is happy to issue “a call for collaboration.” “We cannot work on everything, and we cannot find all the targets to edit,” he notes. “But we have the knowledge to design the vectors and the payloads to make it happen.”

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“We sought to gain insight into a fundamental question: How does genetic variation determine our ‘chemical individuality’—the inherited differences that make us biochemically unique?” said Eric Gamazon, PhD, associate professor of medicine in the Division of Genetic Medicine at Vanderbilt University Medical Center. Gamazon is senior and co-corresponding author of the team’s published report in Nature Genetics, which describes the development of GeneMap and the initial application of the platform.

Kivanç Birsoy, PhD, at the Rockefeller University, is co-senior and co-corresponding author. In their paper, titled “Metabolic gene function discovery platform GeneMAP identifies SLC25A48 as necessary for mitochondrial choline import,” the investigators stated, “… we developed the GeneMAP platform for discovery of metabolic gene function that leverages genetic models of gene expression and quantifies the gene-mediated genetic control of metabolites.”

Metabolic reactions play critical roles in nutrient absorption, energy production, waste disposal, and synthesis of cellular building blocks including proteins, lipids, and nucleic acids, the authors explained. “Given these critical processes, approximately 20% of protein-coding genes are dedicated to maintaining the intracellular chemical landscape and include small-molecule transporters and enzymes.” And while decades of research have revealed the functions of many of these genes, “the exact molecular substrates for many metabolic components remain elusive.”

Abnormalities in metabolic functions are associated with a range of disorders including neurodegenerative diseases and cancers. But, as Gamazon explained, “Despite decades of research, many metabolic genes still lack known molecular substrates. The challenge is in part due to the enormous structural and functional diversity of the proteins.” The authors continued, “Such gaps in our understanding arise partly from diverse tissue-specific expression patterns, functional redundancies, and the metabolic promiscuity of these elements, complicating efforts to define their precise physiological roles.”

The researchers developed the GeneMap platform to discover functions for orphan transporters and enzymes—proteins with unknown substrates. They used datasets from two independent large-scale human metabolome genome-wide/transcriptome-wide association studies (GWAS) and demonstrated with in silico validation that GeneMAP can identify known gene-metabolite associations and discover new ones. They explained, “To identify gene–metabolite relationships, we conducted transcriptome-wide association studies (TWAS) in two independent genomic studies of the human metabolome from the Canadian Longitudinal Study on Aging (CLSA) and the Metabolic Syndrome in Men (METSIM) Study.” In addition, they showed that GeneMAP-derived metabolic networks can be used to infer the biochemical identity of uncharacterized metabolites.

To experimentally validate new gene-metabolite associations, the researchers selected their top finding (SLC25A48-choline) and performed in vitro biochemical studies. SLC25A48 is a mitochondrial transporter that did not have a defined substrate for transport. Choline is an essential nutrient used in multiple metabolic reactions and in the synthesis of cell membrane lipids.

The researchers showed that SLC25A48 is a genetic determinant of plasma choline levels. “Given that SLC25A48 is a member of the SLC25A family that encompasses mitochondrial small-molecule transporters, we hypothesized that SLC25A48 may regulate the availability of choline or its downstream metabolites in mitochondria,” they commented. The investigators then conducted radioactive mitochondrial choline uptake assays and isotope tracing experiments to demonstrate that loss of SLC25A48 impairs mitochondrial choline transport and synthesis of the choline downstream metabolite betaine. “Altogether, our results suggest that SLC25A48 is necessary for mitochondrial choline import and is a key determinant of de novo betaine synthesis in mammalian cells,” they stated.

They also investigated the consequences of the relationship between SLC25A48 and choline on the human medical phenome (symptoms, traits, and diseases listed in electronic health records) using the large-scale biobank resources, UK Biobank and BioVU, Vanderbilt’s DNA biorepository linked to extensive clinical data. These investigations identified eight disease associations.

“What’s exciting about this study is its interdisciplinarity—the combination of genomics and metabolism to identify a long-sought mitochondrial choline transporter,” Gamazon said. “We think, given the extensive in silico validation studies in independent datasets and the proof-of-principle experimental studies, our approach can help identify the substrates of a wide range of enzymes and transporters, and ‘deorphanize’ these metabolic proteins.”

In their paper, the authors concluded, “We developed GeneMAP, a platform for predicting metabolic gene function, and demonstrated its ability to render accurate and replicable results … Because many metabolic enzymes and transporters still do not have identified physiological substrates, GeneMAP provides a unique platform for deorphanizing these genes. This will open up an avenue for understanding the underlying basis of disease as well as development of therapeutics.”

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10x says its Xenium Prime 5K Pan-Tissue and Pathways panel features enhanced chemistry enabling the increase in the number of cells capable of being imaged compared with previous gene expression panels ranging from the company’s 247-gene mouse brain panel to the 379-gene mouse tissue atlassing panel.

“It has a lot of the content that people have asked for from our custom panels,” Ben Hindson, PhD, 10x’s co-founder and CSO, told GEN Edge. “It’s a nice panel in terms of pretty comprehensive coverage of the transcriptome, and it should also enable customers to just take their samples and get lots of really informative data after the run is complete.”

With 5,000 genes, the new panel is designed for comprehensive profiling of cell types and states and cell signaling pathways across multiple tissue and disease types, according to 10x. The panel can be customized with up to 100 additional genes, including isoforms, exogenous sequences such as guide RNAs (gRNAs) and barcodes, chimeric antigen receptor T-cell (CAR-T) transcripts, and viruses.

The breadth of the panel has attracted interest from potential customers within biopharma, as well as academic researchers specializing in single-cell research.

Covers 80% of requests

“I would say it covers about 80% of the requests that we’ve gotten from all those customers that we’ve worked with on either the tissue-specific or the disease-specific panel. It pretty well encompasses a lot of the most common requests, but it also offers customers customization for those who maybe feel that there’s something that’s not quite included that should be.”

What makes the other 20% of requests a no-go for adding to the panel? A combination of feedback from early-access customers and the utility value of the genes: “I would say that some genes aren’t that interesting, and they take up a lot of real estate and don’t give you as much information. So, we try to take those off the list per se, because they’re not very informative.”

The list price for Xenium Prime 5K is $7,500 for each slide. Each run is two slides, making the total cost per run $15,000 in the United States. Xenium Prime 5K also delivers improvements from previous panels that include enhanced per-gene sensitivity, improved specificity and spatial fidelity, integrated multimodal cell segmentation, a workflow compatible with both fresh frozen and FFPE samples—plus the ability to let researchers analyze up to 472 mm² of human or mouse tissue in six days or less—speed, and throughput that the company calls industry-leading.

While Xenium Prime 5K may feature fewer genes than NanoString’s panel, Hindson said 10x has focused heavily on the panel’s performance: “For the most part, we’re pleased with how those landed in terms of sensitivity, specificity, and throughput, together with in-line data analysis and data processing that’s happening while the instrument’s running, so when it’s done,  you get your data and you go off and start analyzing.”

And while numerous research areas can benefit from the panel, Hindson said, the biggest potential opportunity for gleaning knowledge through Xenium Prime 5K is in oncology as researchers delve into the tumor microenvironment, looking at the single-cell resolution, how cells are programmed, how close they are to one another, and how they may interact with each other.

“I think there’s potential for new biomarkers based on the spatiality data as well. I think that’s why at conferences like AACR [the American Association for Cancer Research], there’s just a tremendous amount of excitement for looking at this high content, in situ data for studying the tumor microenvironment and then looking for potential new therapies, looking for why therapies may have worked in one patient and not in the other.”

10x formally announced the launch of the panel in late May, four months after co-founder and CEO Serge Saxonov, PhD, first announced Xenium Prime 5K among a series of product improvements planned for this year in January at J.P. Morgan’s 42^nd Annual Healthcare Conference.

NanoString launches 6K panel

The following month at the annual Advances in Genome Biology and Technology (AGBT) 2024 conference in Orlando, FL, NanoString announced the launch and full commercial release of its CosMx Human 6K Discovery Panel, the first single-cell spatial panel enabling research scientists to measure over 6,000 RNA targets, representing nearly every human biological pathway.

“From the technical perspective, we pushed the envelope harder than anybody else by far,” Joe Beechem, PhD, NanoString’s CSO and senior vice president of research and development, told GEN in an exclusive interview from AGBT.

Human 6K enables analysis of 6,175 genes with 20 control targets, and the ability to customize up to 200 additional targets of interest.

“The applications that we built this panel for were very broad. We went after all of translational biology, with a strong focus on cancer, immunology, and neuroscience. Those are hallmarks for us here at NanoString, the biggest areas of translational research,” Erin Piazza, PhD, associate director of bioinformatics with NanoString, told GEN Edge recently. “But we also looked at many other applications things crossing all of physiology endocrinology and metabolism, cardiovascular disease, developmental biology. We cast a very broad net with this panel, with the hope that most researchers, especially in the translational space, would be able to take advantage of it.”

Beyond numbers

The more genes a panel can accommodate, the better for researchers, though Piazza added that NanoString’s approach goes beyond numbers:

“Researchers love plex. When offered panels of varying size, larger is almost always preferred because it reduces the concern that the researcher has missed important findings. It reveals deeper biological insights and conserves precious tissue samples,” Piazza said.

“While we are delivering to the market the highest plex panels available, our approach is more than sheer numbers,” she said. “Our value proposition to researchers is providing biologically curated high-plex data that meaningfully advances their understanding of a broad variety of translational and discovery research areas.”

NanoString says its approach to content development starts with knowing what genes to include, and which to exclude through a proprietary pruning process, and also includes curating single-cell data based on feedback from key opinion leaders. The company will often share a draft of the genes it wants to feature in a panel under a nondisclosure agreement before locking in a final list, Piazza said.

“If you select the wrong dataset, you make the wrong choices. So we’re very cautious about ensuring that the single cell datasets that we did make decisions based on were as broad as the application areas we wanted to cover,” Piazza explained. “You want to look at normal tissues, disease tissues. What those genes are doing in those contexts informs the decisions you make while you prune. So, there’s a heavy lift of a panel. But we’re really, really pleased with how it turned out.”

NanoString will not disclose the list price for CosMx Human 6K or other products, citing numerous variables. Human 6K is designed for use with NanoString’s CosMx Spatial Molecular Imager (SMI), which offers single-cell RNA and protein imaging with what the company says is the most precise cell segmentation and highest plex available. CosMx SMI also delivers cell typing and spatial context, and accurately measures the transcriptional programs within cells that result in disease or health, according to NanoString.

Piazza said her company’s 6,000-plex panel will someday give way to an even larger panel. During AGBT, NanoString demonstrated what it said was true single-cell whole transcriptome analysis capabilities, releasing the first such public data set.

NanoString expects to make a whole transcriptome panel product commercially available in 2025.

“We’re calling the 6K panel high plex today, but hopefully tomorrow it will be the mid-high plex, as we’ve started already talking about our CosMx whole transcriptome panel,” Piazza said.

The post Aiming High: 10x, NanoString Launch Large-Scale Panels appeared first on GEN - Genetic Engineering and Biotechnology News.

There is surging interest (and competition) in the proteomics industry, reminiscent of the state of NGS and genomics a decade ago. While mass spectrometry (MS) technologies remain the tried-and-true method for analyzing the proteome, established MS companies have been joined by a powerful set of emerging technologies offering the ability to survey more proteins, with higher sensitivity, specificity and greater ease. Indeed, some companies aim to detect disease using protein measurements. This is providing researchers who need to assess the proteome choices that they simply did not have before.

On this episode of GEN Live, we will talk about proteomics—as a tool and as a field. Where is it now and what type of research is proteomics enabling? We will also discuss the latest technologies and instruments in this space. What will it take for these new companies to compete?

A live Q&A session will follow the discussion, offering a chance to pose questions to our expert panelists.

The post Is Proteomics the Next Big “Ome”? appeared first on GEN - Genetic Engineering and Biotechnology News.

Previously, the Karolinska team developed nanostructures capable of housing six peptides assembled in a hexagonal pattern. Under the right conditions, “this hexagonal nanopattern of peptides becomes a lethal weapon,” said Björn Högberg, PhD, a professor in the department of medical biochemistry and biophysics at Karolinska and senior author on the study. That’s because these peptides can “autonomously and selectively turn on the display of cytotoxic ligand patterns in tumor microenvironments” to trigger the apoptosis machinery in cells.

The challenge, however, is that the peptides can also interact with healthy cells so “if you were to administer it as a drug, it would indiscriminately start killing cells in the body,” Högberg explained. “To get around this problem, we have hidden the weapon inside a nanostructure built from DNA.” 

As described in the paper, the origami that the team designed is “an asymmetric double cylinder with a 24-nm-tall hollow head and a 15-nm-tall solid stem. The head has a 14-nm-deep cavity, where specific oligonucleotides … are located to act as bridges between the typical origami staples and a binding site for ligand-decorated oligos.” These oligonucleotide or mini-scaffolds are used to “hybridize a peptide ligand-functionalized oligo, bearing one sequence that forms the double helix (the hybridization region) with the mini-scaffold as well as bearing an additional sequence of triplex-forming oligo (TFO),” the researchers wrote. 

The key to activating the kill switch is the low pH typically found in the acidic microenvironment surrounding cancer cells. Specifically, “when the pH drops, the TFO of the peptide–DNA conjugate forms a tsDNA that forces the six peptides that were originally hidden in the cavity to get displayed as a hexagonal pattern on the top surface of the origami.”

Using cells in test tubes, the researchers demonstrated that the peptide switch remains hidden inside the nanostructure at normal pH. It is activated when the pH drops to 6.5. In fact, “after 24 and 48 h, less than 20% and 10% of cells survived, respectively,” the researchers wrote. “We have managed to hide the weapon in such a way that it can only be exposed in the environment found in and around a solid tumor,” Högberg said. “This means that we have created a type of nanorobot that can specifically target and kill cancer cells.”

Next, the researchers tested the treatment in human breast cancer mouse models. They reported that the treatment resulted in a 70% reduction in tumor growth compared to control mice that received an inactive version of the nanorobot. 

For their next steps, the scientists plan to investigate whether the treatment works in more advanced cancer models that resemble human disease as well as any potential side effects from administering the treatment in humans. They’ll also look into the feasibility of making the nanorobots more precise by attaching proteins or peptides to the structure’s surface that specifically bind to certain cancer types.

The post DNA Origami Selectively Triggers Cytotoxicity to Make Cancer Fold appeared first on GEN - Genetic Engineering and Biotechnology News.

But many single-cell researchers share a common gripe: the experiments are expensive to run. Making the transition from bulk RNA sequencing to scRNA-seq can be a steep challenge for some investigators who must sacrifice sample size to be able to afford it. “Democratization” is a buzz word at single-cell meetings.

There are more than 5,000 Chromium instruments placed around the world, and they are a common feature of university core facilities. But many researchers still have to work hard to find one. For example, GEN heard about a researcher in Bilbao, Spain, who has to drive their samples more than 300 miles to use the nearest Chromium in Madrid. It is fairly common for researchers to bike samples across busy cities or to use the subway to reach a Chromium.

An interesting trend has emerged over the past few years: several newer companies have launched kit-based products that offer single-cell researchers an alternative that doesn’t involve additional instrumentation. Here, GEN relates what several of them have to say about their technology, their products, and their views on the future of single-cell technology.

Go big or go home

“It’s a really exciting time in ‘single cell,’” proclaims Alex Rosenberg, PhD, co-founder and CEO of Parse Biosciences in Seattle, WA. He points to two exciting trends. First, many researchers are entering single cell for the first time. Second, current single-cell users are scaling up. The result is that more groups are utilizing single cell to its full potential.

“Every year,” Rosenberg observes, “the size of projects is doubling with respect to the number of cells people are running.” The decreasing cost of sequencing has helped. As more and more researchers gain single-cell experience, they have realized the value of the technology and are expanding into much larger projects.

In addition, the single-cell community is aware of advances happening in generative AI. There is a big push, Rosenberg notes, to create foundational models for single-cell data that can, for example, predict which cells are going to respond to drug treatment or which cancer cells are going to elicit an immune response. But more data are needed. The largest models have been trained on about 50 million cells. “That’s really not that big in the scheme of things,” he says. In comparison, Open AI trained on data in the range of 10 trillion tokens. People realize that, for now, the single-cell field is data-limited.

Parse considers itself part of the trend in scaling up. One researcher can run a million cells in a single Parse experiment. The company has launched kits to perform B-cell receptor and T-cell receptor profiling—an area where scaling up makes sense given the complexity of the immune repertoire. Parse is also working on making single-cell experimentation easier, both on the front end and the back end.

Data analysis has been challenging. It takes a lot of time and work—even for a bioinformatician. Earlier this year, Parse acquired Biomage, the developer of Cellenics, software that allows single-cell data analysis in a browser without requiring the use of Python or R. That will make data analysis easier for the average biologist. Parse is also enabling automation on its workflow through a partnership with Integra Biosciences. The automation instrument is sold as a bundle with Parse’s kits to ease the pipette-heavy workflow.

Long reads in Rosario

In the middle of the COVID-19 pandemic, a group of scientists in Argentina decided to share their thoughts about the future of single-cell technology. Their first prediction, recalls Elizabeth Tapia, PhD, co-founder and CSO of ArgenTAG, was that the scarcity and inability to access single-cell instruments in South America would persist. Their second prediction was that long reads would gain traction in transcriptomics.

In 2020, ArgenTag was founded by Tapia along with Pilar Bulacio, PhD, Joaquin Ezpeleta, Sofía Lavista Llanos, PhD, and Leandro Ciappina. Today, Bulacio, Ezpeleta, Lavista Llanos, and Ciappina serve the company as CTO, Director of Engineering, Director of Process Development, and CEO, respectively. ArgenTAG has an R&D facility in Rosario, Argentina, and an office in the United States. The company is currently in early access mode and expects to launch its first kit in early 2025.

ArgenTAG introduced its core technology in Scientific Reports in May 2022, in a paper titled, “Robust and scalable barcoding for massively parallel long-read sequencing.” The paper’s authors reported that the technology is designed to enable multiplex long-read sequencing. They also presented evidence that they used the technology to sequence almost 4,000 barcodes simultaneously on Oxford Nanopore Technology’s MinION platform, achieving a high recovery rate and low crosstalk.

“We took the worst-case scenario of long-read accuracy and improved the accuracy until it became comparable to that for an Illumina sequencer,” Ciappina notes. Then they started deploying barcodes in the cells for a single-cell solution.

Why focus on long reads? “When you do short reads, you’re not getting all the information,” Lavista Llanos tells GEN. “You’re just getting a tip of the information.” She adds that short reads provide just a snapshot.

Lavista Llanos has a background in embryogenesis, a discipline in which isoforms are expressed over hours or even minutes. The ability to collect those data to further explore developmental biology is also important in other disciplines. Indeed, according to Lavista Llanos, the folks at ArgenTAG believe that it is the future of single-cell technology. “Our technology makes sense today,” she remarks, “but it will make a lot more sense in five years.”

ArgenTAG’s workflow uses chip-based technology for partitioning and barcoding. Essentially, the chip’s microwells partition the cells and contain beads that carry the barcodes. This technology differs from competing technologies by performing sequencing on an Oxford Nanopore or PacBio instrument instead of an Illumina instrument.

“Our vision is to decentralize single cell today,” Ciappina declares. “10x Genomics has 5,000 instruments installed, but there is a bigger opportunity—100,000 labs that can benefit from single-cell sequencing but cannot today because of the price.”

Génomique unicellulaire

Stuart Edelstein, PhD, a renowned biologist who trained with Nobel laureate Jacques Monod and held posts at Cornell University and the University of Geneva, has been retired for years. But Wilko Duprez, PhD, head of communications at Scipio Bioscience, tells GEN that Edelstein’s mind “never stops thinking about science.” When 10x Genomics released its single-cell, microfluidics-based technology, Edelstein was hiking the volcanoes of Hawaii. Inspired by porous rocks, Edelstein hit upon an idea that would turn out to be an alternative to microfluidics for single cell. “Why can’t we have something that would fit one cell in each little pocket, like the volcanic rock?” Edelstein pondered. The solution was a hydrogel that physically isolates each cell without microfluidics. Edelstein founded Scipio in 2017 and recruited scientists to bring his idea to fruition.

In May 2022, Scipio launched its first kit, Asteria, which can process up to four samples, each with 15,000 cells. The low throughput may appeal to researchers who want to dabble in single cell or have a low number of samples. The only hydrogel kit on the market, Asteria does not manipulate the cells in any way: no fixing, freezing, sorting, vortexing, etc. Duprez says that cells are “happy and cushioned sitting in the hydrogel.”

How does it work? A single-cell suspension is mixed with a bead solution to form cell-bead pairs with one barcoded bead per cell. In a 50 mL tube, the solution is diluted into the hydrogel, which is in a liquid state. A piston-like tool is inserted to ensure there is a thin, uniform layer of hydrogel spread around the walls of the tube. Once put on ice, the hydrogel solidifies with cell-bead pairs isolated into pockets. After the addition of lysis buffer, the mRNA is captured by a bead. The hydrogel is thawed back to liquid using a buffer (the hydrogel doesn’t turn to liquid when it warms up), and the beads are recovered. From there, the standard RNA-seq protocol is followed starting with on-bead reverse transcriptase and PCR. The Asteria kit does not include library prep at the moment, but Scipio plans to include it in the future.

Scipio is located at the Cochin Hospital in Paris, where it has access to clinical samples. The hydrogel is well suited for multisite clinical studies because it is very stable for a long time (at least a month at −80°C). Some clinical studies are multisite and include hospitals or clinics in different locations. Because transcriptomics is time-sensitive, if there are multiple centers collecting samples for single-cell analysis using a kit is easier than having an instrument in five different locations. Hydrogels could be shipped to the same place so that sequencing can be done together. In addition, the Asteria kit can now accommodate CITE-seq data by incorporating tagged antibodies measuring the presence of surface proteins at the single-cell level, therefore achieving multiomics data within a single experiment

Moving into the clinic

The core technology at Fluent BioSciences—pre-templated instant partition sequencing (PIPseq)—originated in the laboratory of Adam Abate, PhD, professor of bioengineering and therapeutic sciences at the University of California, San Francisco. More than a decade ago, Abate and Sepehr Kiani, PhD, chief business development officer at Fluent, started Gnubio (later sold to Bio-Rad Laboratories in 2014), which had developed a technology to inject femtoliter volumes into droplets. In 2018, Abate told Kiani about a new tool he had been working on. Kiani tells GEN that the tool was designed to “do everything RainDance Technologies [another company acquired by Bio-Rad] could do but without any instrumentation.” Once Abate’s laboratory established proof of concept, PIPseq technology attracted investment and led to the founding of Fluent Biosciences in 2020.

The company launched its first kit in July 2022. After five iterations of PIPseq technology, the PIPseq V launched in May 2024. About 30% of Fluent’s customers are newcomers to the field. “We’re really about enabling single cell,” Kiani says. According to Kiani, colleagues of his have met many scientists who are interested in single cell but have shied away because it is too expensive, too complicated, or demands instrumentation they lack.

In the PIPseq kit, a tube has hundreds of thousands (or even millions) of template particles—organic polymers that define the partition and carry a barcode. Fluent sells kits that can manage two to eight reactions and collections of cells that range from a few thousand to hundreds of thousands (with one million cells in early access).

One advantage of Fluent’s technology is that researchers can use it to interrogate the transcriptome multiple times. Researchers can employ the standard method (which sequences the three-prime end), reanalyze the cells for targeted transcriptomics on the same cells, and then go back to perform long reads. Researchers can also process many cells in one sample, which speaks to Fluent’s vision of moving into clinical applications in the future. This large-scale per sample workflow is aligned with clinical utility, Kiani notes. “Fluent is going to own the clinic,” he predicts. “It’s the only thing with the elegance to go into the clinic.”

Other companies introducing single-cell technology in kits include Scale Biosciences, which is located in San Diego, CA, and Singleron Biotechnologies, which has offices in Michigan and Connecticut, as well as in Germany, Singapore, and China. Scale was co-founded by Jay Shendure, PhD, professor of genome sciences at the University of Washington, Cole Trapnell, PhD, associate professor of genome sciences at the University of Washington, and Garry Nolan, PhD, professor of pathology at Stanford University. The company offers multiple kits: a single-cell RNA kit, a single-cell ATAC kit, and a single-cell methylation kit. (One customer told GEN that the methylation kit, in particular, is exciting and sets Scale apart.)

Singleron was founded by Nan Fang, PhD, formerly of QIAGEN and Novogene, and Jing Zhou, PhD, formerly of Dilworth IP and IsoPlexis. Singleron uses a SCOPE-chip microwell microfluidics system that uses gravity to guide cells into wells and is designed for gentle single-cell partitioning.

Regardless of the technology, the company, or the kit, there are an increasing number of options for researchers interested in obtaining gene expression data of single cells. The consensus is that the field is just beginning. Current technologies can reach about 20% of the roughly 250,000 mRNA transcripts in a mammalian cell. The issue is not the abundance of molecular probes. There are enough barcodes to label every mRNA. So, what is the limitation? Hydridization, reverse transcription, mRNA secondary structure, or library complexity? Opinions vary. But given the momentum in the single-cell community, it won’t be long before limitations are overcome.

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